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Introduction

Islet transplantation is a promising treatment for patients with type 1 diabetes mellitus who frequently have severe hyperglycemia and glycemic instability (1,2). Islet transplantation using the Edmonton protocol can successfully restore long-term endogenous insulin production and glycemic stability in patients with type 1 diabetes mellitus (3). The administration of immunosuppressive therapy that causes less damage to pancreatic islet cells is a critical consideration. Before the Edmonton protocol, immunosuppressants, such as tacrolimus, cyclosporine, azathioprine, glucocorticoids, and antilymphocyte globulin were used similar to other organ transplantation. Because glucocorticoids were found to enhance insulin resistance, while tacrolimus and cyclosporine were observed to suppress insulin secretion and cause kidney damage, the Edmonton protocol introduced a new immunosuppressive therapy using sirolimus and anti-interleukin (IL)-2 receptor antibodies (daclizumab) with low-dose tacrolimus. However, the results of the Edmonton protocol at that time were unsatisfactory with a 5-year insulin withdrawal rate of 7.5% (4). The Collaborative Islet Transplant Registry (CITR) protocol with anti-thymocyte globulin and anti-tumor necrosis factor (TNF) α antibody (etanercept) instead of anti-IL-2 receptor antibodies, further improves islet transplant outcomes (1,5). Reportedly, the 4-year graft survival rate was approximately 90% for transplant cases between 2007 and 2010, and the 3-year insulin withdrawal rate was 44%.

In 1991, Izumori (6) discovered a new enzyme for synthesizing rare sugars and established a method to mass produce rare sugars. According to the International Rare Sugar Association, monosaccharides and their derivatives are rare in nature. D-allose, the C3 epimer of D-glucose, is a rare sugar produced from D-fructose that has various antioxidant, anti-inflammatory, and immunosuppressive effects (7,8). Previous reports (8,9) indicate that D-allose is immunosuppressive without toxic side effects, unlike FK506 (tacrolimus), as D-allose induced a dose-dependent suppression of segmented neutrophils. In liver transplantation, prolonged liver allograft survival in rats can be achieved using a combination of FK506 and D-allose than either drug individually (8). D-allose has been shown to have a protective effect owing to its anti-inflammatory and immunosuppressive effects, and antioxidant activity against ischemia-reperfusion injury in several organs such as the liver (9), brain (10,11), and skin (12,13).

Based on accumulated evidence of the antioxidant effects of D-allose in transplanted cells, we previously focused on improving insulin secretion after transplantation of pancreatic islets. We demonstrated that D-allose treatment of isolated islet cultures prior to transplantation restored islet function and increased transplant success rates (14). D-allose has been suggested to improve the function of damaged islets through its antioxidant activity.

In the present study, we further examined whether intravenous D-allose administration could improve insulin secretion when pancreatic islets were transplanted into type 1 diabetes model mice. This would suggest that D-allose is useful both as an immunosuppressant in human pancreatic islet transplantation and as a protective agent against cell damage through its anti-inflammatory effects. Furthermore, D-allose is safe enough to be administered intravenously, therefore it may be possible to implement it quickly in clinical settings.

Materials and methods

Animals

Male BALB/c mice aged 8-12 weeks purchased from CLEA Japan, Inc. (Tokyo, Japan) and Japan SLC, Inc. (Shizuoka, Japan) were used for all the experiments. All mice were bred and handled according to the Experimental Animal Research Guide. This study was approved by the Animal Care and Use Committee of Kagawa University. Since only male mice were used in our previous study (14), we conducted experiments using male mice as a continuation of the previous study in this study. A total of 60 mice were used as a mouse model of type 1 diabetes by STZ induction, and 138 healthy mice were used for islet extraction for islet transplantation. The handling of euthanasia of mice used in this study was performed according to the AVMA guidelines for the euthanasia of animals (2013 edition) (15). Animals will be closely monitored after treatment and will be euthanized if they have difficulty feeding or ingesting water, show rapid and unrecoverable weight loss (>25% in 7 days), or show signs of weakness such as abnormal posture, breathing problems, bleeding or anemia, or if the experiment is stopped and the animal is euthanized. Healthy mice whose pancreas was removed to extract the islets were sacrificed by severing the vena cava. The mice with one kidney removed, from which the islets were transplanted, were euthanized with carbon dioxide within a week because they became hyperglycemic and debilitated. Euthanasia by carbon dioxide was carried out in a designated area of the animal experimental facility. The carbon dioxide replacement was performed at a rate of 30%/min at our facility. In detail, mice were placed in cages and 100% CO2 gas was flowed from a compressed carbon dioxide gas cylinder at a flow rate of 5 l/min for 5 min. During this time, the mice stopped breathing and their eyeballs became discolored. After 5 min, the flow of CO2 was stopped, the container was sealed, and more than 5 min were allowed to pass. Death was then confirmed by palpation to check the heartbeat.

Creation of diabetic mice

Streptozotocin (STZ) 200 mg/kg body weight (BW) was administered via the tail vein to prepare type 1 diabetes model mice. A diabetic mouse model was defined as one in which the casual blood glucose level was >400 mg/dl for >2 consecutive days. Blood was collected by making a small incision in the skin at the tip of the mouse's tail. Less than 1 µl of blood is needed to measure blood glucose, so a little more than this was collected.

Islet isolation and culture method

Under inhalation anesthesia with isoflurane (introduction: O2 0.5L, isoflurane 4-5%, maintenance: O2 0.5L, isoflurane 2-3%) or sevoflurane (introduction: O2 0.5L, sevoflurane 5%, maintenance: O2 0.5L, isoflurane 2.5-4%), depending on the anesthesia machine, cold Hanks' Balanced Salt Solution (HBSS; Sigma, St. Louis, MO, USA) containing dissolved collagenase (collagenase type V; Wako Pure Chemical Industries, Ltd., Osaka, Japan) at a concentration of 2 mg/ml (0.2%) was injected through the common bile duct under microscope, to dilate the pancreas, which was then removed. The pancreas was digested by warm bathing at 37˚C for 25 min. Subsequently, concentration gradient centrifugation was performed using Histopaque 1077 (Sigma) and HBSS, and the solution layer containing the islets was recovered. Then, the islets were cultured with RPMI1640 medium (Sigma) supplemented with 5.6 mmol/l glucose and 5% fetal bovine serum at 37˚C and 5% CO2 for 2 h.

Islet transplantation and examination of blood glucose level

Transplantation of 15 islet equivalents (IEQ)/g (per BW of recipient mice) of pancreatic islets using islets extracted from two healthy mice was performed under the capsule of one of kidneys in each diabetic model mouse. The method of transplantation under the renal capsule followed our previous study (14). Of the 19 diabetic model mice transplanted with pancreatic islets, D-allose, which was supplied by the Kagawa University Rare Sugar Research Center (Kagawa, Japan), was administered to 9 mice (D-group), while physiological saline was administered to 10 mice as the control group (C-group) (Fig. 1A). D-allose (400 µg/g BW) and physiological saline were intravenously injected in the tail vein three times (0, 24, and 48 h after transplantation). Dose concentrations of D-allose were determined according to previous papers reported from our institution (16,17).

Figure 1 (A) Schematic representation of D-allose administration. (B) Comparison of casual blood glucose levels between the D- (n=9) and C-groups (n=10). Casual blood glucose levels in mice were examined at several time points: Before treatment with streptozotocin (before transplantation), before transplantation of pancreatic islets after treatment with streptozotocin (0 day), at 7, 14 and 21 days, and the day before and after extraction of transplanted islets (prior to harvest and post-harvest, respectively). There was a significant difference in casual blood glucose levels between the D- and C-groups at 14 and 21 days (*P=0.038 and **P=0.025, respectively). BW, body weight; C-group, mice that received physiological saline as a control; D-group, mice that received an intravenous injection of D-allose.

All mice were permitted ad libitum access to food and water during this study. Casual blood glucose levels of the mice were examined at several time points (before treatment with streptozotocin, transplantation of pancreatic islets after treatment with streptozotocin (0 day), 7, 14, 21 days, and the day before and after extraction of transplanted islets).

IPGGT

Five mice each from the D-group and C-group, were prepared in the same manner as above and fasted for 12 h overnight on the seventh day after transplantation (Fig. 2A). Then, IPGGT was performed. Blood glucose was monitored at various time points (0, 15, 30, 60, 90, and 120 min) after intraperitoneal injection of glucose solution (2 g glucose/kg BW) by cutting the tail and gently massaging the blood onto a glucose test strip.

Figure 2 (A) Flow chart of the experiment for the intraperitoneal glucose tolerance test. (B) Blood glucose levels were monitored at various time points (0, 15, 30, 60, 90 and 120 min) after intraperitoneal injection of glucose in the D- (n=5) and C-groups (n=5). There were significant differences in the blood glucose levels between the D- and C-groups at 30, 60, 90 and 120 min (*P=0.0066, **P=0.0054, ***P=0.01 and ****P=0.01, respectively). (C) IAUC of blood glucose concentration. The IAUC of glucose was significantly decreased in the D-group compared with the C-group (P=0.0054). BW, body weight; C-group, mice that received physiological saline as a control; D-group, mice that received an intravenous injection of D-allose; IAUC, incremental area under the curve.

The incremental area under the curve (IAUC) of blood glucose concentration was calculated using the method described by Wolever and Jenkins (18).

Histological examination of the effects of D-allose administration into recipients

To evaluate apoptotic islet cells, 30 IEQ/g (per BW of recipient mouse) of pancreatic islets was transplanted under the bilateral renal capsule of non-diabetic BALB/c mice. Transplantation was performed in six mice in the D-group and six mice in the C-group (Fig. 3A). Mice in the D-group were intravenously injected 400 µg/g BW of D-allose dissolved in physiological saline, through the tail vein, 24 and 48 h after transplantation. Mice in the C-group were intravenously injected with physiological saline. On the 3rd postoperative day, the islet-transplanted kidney was removed. The excised right kidney was fixed in formalin, and the left kidney was immediately stored at -80˚C.

Figure 3 (A) Flow chart of the experiment up to nephrectomy for TUNEL staining. (B) Representative TUNEL staining images. (B-a) H&E staining of islet cells transplanted and engrafted under the renal capsule in the C-group (magnification, x40). (B-b) TUNEL staining of the enlarged white square area in B-a. Only one positive cell was observed (white arrow). Scale bar, 100 µm. (B-c) H&E staining of islet cells transplanted and engrafted under the renal capsule in the D-group (magnification, x40). (B-d) TUNEL staining of the enlarged white square area in B-c. Two positive cells were observed (white arrows). Scale bar, 100 µm. BW, body weight; C-group, mice that received physiological saline as a control; D-group, mice that received an intravenous injection of D-allose.

The kidney tissue samples of each group were fixed in 4% paraformaldehyde for 48 h. The sample was embedded in paraffin wax and cut into 4 µm slices. After defatting, the sections underwent hematoxylin and eosin staining, followed by dehydration and sealing. The stained sections were observed under a microscope (Olympus, Japan).

Apoptotic cells were examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, using an In Situ Apoptosis Detection Kit (MK-500; Takara Bio Inc., Tokyo, Japan) according to the manufacturer's instructions. Briefly, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Endogenous peroxidases were inactivated by incubating the cells in 3% hydrogen peroxide for 5 min. After TUNEL staining with TdT enzyme for 90 min at 37˚C in the dark, sections were treated with streptavidin-HRP, and apoptotic cells were visualized using DAB staining, resulting in brown staining. Hematoxylin was used for counterstaining. TUNEL-positive cells were examined under a microscope. Three fields were randomly selected for each sample, and the number of apoptotic cells was calculated.

Reverse transcription-quantitative PCR (RT-qPCR)

The gene expression of caspase 3 (CASP3), heme oxygenase 1 (HMOX1), and nitric oxide synthase 2 (NOS2) in engrafted cells with or without D-allose administration, was examined using RT-qPCR. Six diabetic model mice in both the D- and C-groups received islet transplants (Fig. 4A).

Figure 4 (A) Flow chart of experiments up to nephrectomy for gene mRNA expression analysis. (B) Comparison of CASP3, HMOX1 and NOS2 mRNA expression between the engrafted cells of mice in the D- (n=6) and C-groups (n=6). The mRNA expression of each gene was normalized to that of ACTB. The expression level of each gene was shown as a relative ratio to the D-group, with the gene expression level in the C-group set to 1. BW, body weight; C-group, mice that received physiological saline as a control; D-group, mice that received an intravenous injection of D-allose. CASP3, caspase 3; HMOX1, heme oxygenase 1; NOS2, nitric oxide synthase 2.

In the D-group, 400 µg/g BW of D-allose dissolved in saline was intravenously administered via the tail vein immediately after transplantation and 24 h after transplantation. In the C-group, saline alone was administered intravenously. After 1 h, the transplanted islets were extracted alone with the surrounding renal parenchyma under anesthesia. The extracted tissue was immediately stored at liquid nitrogen temperature, and subsequently at -80˚C.

Bead-type cell disruption was performed on the excised tissue, and RNA was extracted using the spin column method. For RNA, contamination with impurities was confirmed by absorbance measurement (NanoDrop 2000, Thermo Fisher Scientific), and RNA concentrations of >100 ng/µl, A260/280 ratio >1.8, and A260/230 ratio >1.5 were accepted. cDNA was synthesized from the extracted RNA by reverse transcription using the PrimeScript RT Master Mix (Takara Bio). The amount of RNA was adjusted such that the total RNA was 500 ng/2 µl of the absorbance PrimeScript RT Master Mix. qPCR was performed to examine the mRNA expression of CASP3, HMOX1, NOS2, and actin beta (ACTB) using TaqMan probe Mm01195085_m1, Mm00516005_m1, Mm00440502_m1, and Mm02619580_g1 respectively (Thermo Fisher Scientific) on the Applied Biosystems real-time PCR system ViiA ™ 7. The qPCR conditions were applied according to the manufacturer's instructions. The mRNA expression level of each gene was normalized to that of ACTB, and the expression levels were analyzed using the 2-ΔΔCq method (19).

Statistical analysis

All values are expressed as means ± standard error (SE). Comparisons of glucose values at each timepoint, IAUC, the number of engrafted cells and mRNA expression levels between the two groups were statistically analyzed using an unpaired t-test. Statistical significance was set at p<0.05.

Results

Improvement of casual blood glucose level in D-allose-administered mice

The average ± SE of casual blood glucose level in the D-group (n=9) at 0, 7, 14, 21, and 28 (prior to harvest) days was 446.67±14.7, 113.56±16.71, 100.44±5.58, 100.89±4.29, and 109.44±6.7 mg/dl, respectively, while that in the C-group (n=10) at 0, 7, 14, 21, and 28 days was 465.7±20.78, 196.1±37.83, 232.7±55.32, 236.4±52.07, and 205±42.98 mg/dl, respectively (Fig. 1B). There was a significant difference in casual blood glucose levels between the D- and C-groups at 14 and 21 days (P=0.038 and 0.025, respectively).

IPGGT

After intraperitoneal injection of glucose solution, the average ± SE of the blood glucose level in the D-group (n=5) at 0, 15, 30, 60, 90, and 120 min was 55.8±2.96, 200.8±16.17, 211±5.08, 140±18.21, 88.6±10.39, and 67.8±8.97 mg/dl, respectively, while that in the C-group (n=5) at 0, 15, 30, 60, 90, and 120 min was 115.2±21.11, 279.6±31.24, 356±39.47, 330.6±47.06, 246±45.8, and 178±31.82 mg/dl, respectively (Fig. 2B). There was a significant difference between blood glucose levels in the D- and C-groups at 30, 60, 90, and 120 min (P=0.0066, 0.0054, 0.01 and 0.01, respectively).

Based on the alteration of glucose in IPGGT, IAUC of glucose in the C- and D-groups was 321.2±40.22 and 158.53±15.46 mg-h/dl, respectively (Fig. 2C). The IAUC of glucose was significantly lower in the D-group than that in the C-group (P=0.0054).

HE and TUNEL staining to evaluate apoptosis

There was no difference in the morphology of the transplanted islets under pathological staining between the control and D-allose groups (Fig. 3B).

The representative TUNEL staining images were shown in Fig. 3. The mean number of engrafted cells in the C- and D-groups was 469.8±123.2 and 479.67±84.14, respectively (Table I). No statistically significant difference was observed in the number of engrafted cells between the groups (P=0.95). Among them, few apoptotic cells were observed in both the groups (Table I).

Table I Number of engrafted cells and apoptotic cells in the C- and D-groups.

Table I

Number of engrafted cells and apoptotic cells in the C- and D-groups.

	C-group							D-group
	Case							Case
Cells	1	2	3	4	5	6	Mean	1	2	3	4	5	6	Mean
Engrafted cells	264	332	1,016	560	473	174	470	847	404	529	503	332	263	480
Apoptotic cells	0	1	2	2	1	0	1	3	0	0	1	2	0	1

[i] C-group, mice that received physiological saline as a control; D-group, mice that received an intravenous injection of D-allose.

CASP3, HMOX1, and NOS2 mRNA expression in the engrafted cells

The gene expression associated with apoptosis (CASP3) and cellular damage (HMOX1 and NOS2) was examined to determine how D-allose maintains insulin secretion in engrafted cells. The expression level of each gene is shown as a relative ratio to that of the D-group, with the gene expression level in the C-group set to 1. NOS2 mRNA expression (ratio: 2.06) in the engrafted cells of the D-group tended to be higher than that of the C-group (P=0.07) (Fig. 4B). CASP3 (ratio: 1.26) and HMOX1 (ratio: 1.1) mRNA expression was not significantly different in the engrafted cells between the C- and D-groups.

Discussion

In the present study, we found that intravenous D-allose administration at the time of islet transplantation effectively improved type 1 diabetes mellitus in mice. D-allose is manufactured from D-allulose, which is already in the food supply as a low-calorie sweetener. Although D-allose has not been confirmed to exist in nature until now, it has recently been found in umbilical cord blood (20). Among rare sugars, D-allulose (also known as D-psicose) has a hypoglycemic effect and is expected to be useful in the prevention of type 2 diabetes (21,22). D-Allulose has been reported to dose-dependently suppress blood glucose levels after glucose loading in the OGTT of diabetic volunteer subjects in clinical trials (21). In addition, animal studies have shown that D-allulose was a glucagon-like peptide-1 (GLP-1) releasing substance that limits feeding and hyperglycemia through the vagus nerve (22). On the other hand, D-allose is expected to be useful in the treatment of type 1 diabetes because of its usefulness in maintaining cells following islet transplantation, as in this study, because of its antioxidant and anti-inflammatory effects as previously reported (7,8).

We first examined casual blood glucose levels in type 1 diabetes mellitus model mice treated with D-allose or physiological saline. Mice treated with D-allose showed improved diabetes mellitus status since the casual blood glucose level was <150 mg/dl, which was defined as the normal blood glucose level in BALB/c mice (23), while the casual blood glucose level in untreated mice remained >200 mg/dl on average. Although pancreatic islet transplantation decreased blood glucose levels, the intravenous injection of D-allose was more effective in decreasing blood glucose levels. Moreover, blood glucose levels remained low for more than 21 days with three rounds of intravenous D-allose administration at the time of islet transplantation, suggesting that the D-allose suppressed the decline in insulin secretion during the initial engraftment stage.

Further, we found that D-allose significantly improved diabetes mellitus, based on the results of IPGGT. Changes in blood glucose levels in mice treated with D-allose were lower than those in untreated mice. Consequently, the IAUC of glucose in D-allose-treated mice was significantly lower than that in untreated mice. This result suggests that insulin secretion was rapidly induced by glucose absorption in D-allose-treated mice.

In the present study, pancreatic islet cells were transplanted under the renal capsule in mice, which differs from the transplantation method used in humans. However, D-allose did not cause an increase in the number of grafts but rather prevented a decline in insulin secretion. Notably, no change in the number of transplanted cells was observed with or without D-allose, and no cells underwent significant apoptosis, as determined using TUNEL staining and CASP3 expression. Therefore, the fact that blood glucose levels were kept low by adding D-allose suggests that D-allose maintains the insulin secretion ability of the cells, which may also contribute to the extension of the insulin withdrawal period after transplantation into the human body. We previously demonstrated that D-allose treatment of isolated islet cultures prior to transplantation restored islet function and increased transplant success rates (14). Although the steps involved in D-allose treatment were different, the results were consistent in that no effect on apoptosis was found, but high insulin secretion ability was observed. Considering the results of this study and previous experiments, it is expected that insulin secretion can be further maintained by transplanting islet cells incubated overnight in a medium containing D-allose, and intravenous injection of D-allose after transplantation.

Insulin secretion in transplanted cells may be affected by inflammatory responses to the engrafted cells and immunosuppressants against autoimmunity. Since it is known that D-allose has both anti-inflammatory (10,11) and immunosuppressive effects (8), intravenous D-allose administration would be a promising treatment for pancreatic islet transplantation. In clinical practice, pancreatic islets are transplanted into the liver via the portal vein in humans. According to a study using positron-emission tomography combined with computed tomography (24), approximately half of the pancreatic islets transplanted into the portal vein disappeared immediately after transplantation due to inflammatory reactions and thrombosis. Therefore, suppressing the inflammatory response that occurs during islet transplantation is thought to be key for improving the outcomes. There are many reports on the anti-inflammatory effects of D-allose, including its ability to suppress the expression of cytokines and chemokines involved in inflammation. A previous study (10) demonstrated that intravenous D-allose administration has potent neuroprotective effects against acute cerebral ischemia/reperfusion in a rat model of transient middle cerebral artery occlusion. Another report (11) showed that D-allose administration repressed the levels of TNF-α, nuclear factor kappa B (NF-κB), IL-1β, and IL-8 in inflammatory responses in a mice model of ischemia reperfusion injury. In another model of ischemia reperfusion injury in skin flap, D-allose decreased monocyte chemoattractant protein-1 (MCP-1), TNF-α, IL-1β, and IL-6 levels in the injured skin flap. There appears to be a consensus regarding the anti-inflammatory effect of D-allose, and this effect is potentially involved in maintaining insulin secretion rather than in cell transplantation. Regarding immunosuppressants for autoimmunity during transplantation, changes in clinical protocols have allowed for the extension of insulin withdrawal periods. Treatment with sirolimus and tacrolimus, used in the Edmonton protocol, abolishes beta-cell regeneration, leading to a decrease in insulin secretion (25). The CITR protocol with anti-thymocyte globulin and anti-TNFα antibody has improved islet transplant outcomes (1,5). Previous reports (8,9) indicated that D-allose has an immunosuppressive capability without toxic side effects unlike FK506 (tacrolimus), as D-allose induced a dose-dependent suppression of segmented neutrophils. In liver transplantation, prolonged liver allograft survival in rats was achieved by a combination treatment with FK506 and D-allose than either drug individually (8). The results of the present study suggest that D-allose could be useful in maintaining insulin secretion even in pancreatic islet transplants because of its potential anti-inflammatory and immunosuppressant effects.

We investigated gene expression related to cellular damage to determine how D-allose preserves insulin secretion in engrafted cells. The HMOX1 expression related to heme-mediated oxidative stress was also examined. Heme released from heme proteins amplifies the production of reactive oxygen species (ROS), which are extremely harmful to the body. HMOX1 is the rate-limiting enzyme in heme degradation, and protects the body from heme-mediated oxidative stress (26). The induction of HMOX1 expression in gastrointestinal tissues and cells, including the pancreas, plays a critical role in cytoprotection and resolving inflammation (27). However, our results showed that HMOX1 mRNA expression was not significantly different between engrafted cells in the C- and D-groups, indicating that the engrafted cells were probably not subject to strong oxidative stress. In contrast, NOS2 mRNA expression in the engrafted cells of the D-group tended to be higher than that in the C-group. When exposed to inflammatory cytokines such as IL-1β, β-cells express NOS2, the inducible isoform of nitric oxide (NO) synthase (iNOS). Although previous reports (28,29) have shown the inhibitory effects of NO on insulin secretion, inhibition of NOS decreased insulin secretion from isolated human pancreas (30) and plasma insulin level in healthy humans (31). These conflicting results are reportedly due to the varying concentrations of NO (32). According to a previous study (32), NO at tens of nanomolar concentrations facilitates glucose-induced Ca2+ concentration in β-cells and insulin secretion in a cGMP-dependent manner, whereas NO at sub-micromolar concentrations inhibits them in a cGMP-independent manner. Considering this mechanism, although no significant differences were observed in our results, it is thought that the slight increase in NOS2 expression caused by the addition of D-allose affected the stimulation of insulin secretion. However, the direct function of D-allose was not well understood, although inhibition of inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and inhibition of apoptosis have been reported. Recently, D-allose has been shown to protect the brain from ischemia reperfusion-induced apoptosis and inflammation by suppressing Galectin-3 expression and transcriptional processes that inhibit TLR4 and activate PI3K/AKT phosphorylation (33). In the present study, a hypoglycemic effect was observed in the D-allose-treated group. These results suggest that insulin secretion was promoted by D-allose, which has an inhibitory effect on inflammatory cytokines induced by islet transplantation.

Since islet transplantation-induced inflammatory cytokines suppress insulin secretion (34), D-allose may promote insulin secretion by suppressing the expression of inflammatory cytokines through some molecules. In the future, we would like to clarify the molecules on which D-allose acts directly.

The following limitations exist for this study. First, as a continuation of the previous study, only male mice were analyzed in this study. The reason for using males is that males are more suitable than females in the generation of STZ-induced type 1 diabetes model mice since testosterone enhances the pancreatic β-cell toxicity of STZ (35,36). In the creation of STZ-induced type 1 diabetic mice, 7 of total 60 (11.7%) died of complications due to hyperglycemia within one week. Although the mortality rate was higher than the 4.38% described in the previous report (37), it was possible that the original condition of the mice may have been a factor. Although males have been used in studies with rare sugars, it is possible that different sexes and strains may respond differently. Considering such differences in humans, we would like to continue our research including females in the future. Secondly, in the present study, islets were transplanted under the renal capsule in mice, since the kidney subcapsular site is in close proximity to abundant renal parenchymal blood flow, and it is less susceptible to invasion from the surrounding tissues, making it difficult for the transplanted islets to be washed away or migrate elsewhere. In addition, we had already established the technique to transplant islets under the renal capsule as shown in the previous report (14). However, in humans, islets are clinically transplanted into the liver via the portal vein. According to the previous report (38), islet transplantation under the renal capsule in humans has been reported to have several drawbacks, including the need for a large number of islets and the time required for islet engraftment. Recently, clinical trials have been conducted using induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) to produce islet sheets for subcutaneous transplantation into the abdomen, and favorable results have been reported (39). It is possible that the effect of D-allose may differ depending on the difference in transplantation methods and animal species. In animal experiments, D-allose was administered to healthy mice for a long period of time, and blood tests showed no adverse effects on liver or kidney function (40). Furthermore, it has been reported that long-term oral administration of D-allose improved the intestinal microflora of aged mice (41), indicating that D-allose is safe in animal studies. Although D-allose is considered to be a promising rare sugar for treatment with islets transplantation in animal experiments, no clinical trials in humans have been conducted so far. In the future, it will be necessary to consider what route and how much of D-allose should be administered to humans.

Third, in estimating the reason for the glucose suppression of D-allose in the present results, we did not examine insulin measurements in the kidney and islet samples. We will take this into account when we conduct similar experiments in the future to confirm the effect of D-allose.

In conclusion, we found that intravenous D-allose administration at the time of islet transplantation effectively improved hyperglycemia and maintained stable blood glucose levels in type 1 diabetes mice. Since there was no difference in the number of engrafted cells or apoptotic cells with or without intravenous D-allose administration, D-allose was considered to be effective in maintaining the cellular function of insulin secretion.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

SN, KK, MO, YS and KO conceived and designed the study. SN, HM and YA performed mouse experiments and collected data. SN, HS, AK and TK analyzed the collected data. SN, KK, YS and KO interpreted the results and wrote the manuscript. KK and KO confirm the authenticity of all the raw data. All authors reviewed the results. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

All mice were bred and handled according to the Experimental Animal Research Guide. The present study was approved by the Animal Care and Use Committee of Kagawa University (approval nos. 19646 and 19646-1; Kita-gun, Japan).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References