2024 年 47 巻 4 号 p. 791-795
The number of patients with type 2 diabetes is increasing worldwide. The mechanisms leading to type 2 diabetes and its complications is being researched; however, the pathological mechanisms of diabetes in the small intestine remain unclear. Therefore, we examined these pathological mechanisms in the small intestine using a mouse model of type 2 diabetes (KK-Ay/TaJcl) aged 10 and 50 weeks. The results showed that diabetes worsened with age in the mice with type 2 diabetes. In these mice, advanced glycation end products (AGEs) in the small intestine and mast cell expression increased, whereas diamine oxidase (DAO) decreased; increased tumor necrosis factor (TNF)-α and histamine levels in the plasma and small intestine were also detected. Additionally, the expression of zonula occludens (ZO)-1 and Claudin1 and cell adhesion molecules in the small intestine reduced. These results exacerbated with age. These findings indicate that type 2 diabetes causes AGE/mast cell/histamine and TNF-α signal transmission in the small intestine and decreases small intestinal wall cell adhesion molecules cause TNF-α and histamine to flow into the body, worsening the diabetic condition. In addition, this sequence of events is suggested to be strengthened in aged mice with type 2 diabetes, thus exacerbating the disease. These findings of this study may facilitate the elucidation of the pathological mechanisms of type 2 diabetes and its associated complications.
Type 2 diabetes is caused by the decreased secretion and abnormal action of insulin because of lifestyle changes, accounting for more than 90% of patients with diabetes. This disease often develops after the age of 40 years and the number of patients increase with age.1) The formation of advanced glycation end products (AGEs) is a mechanism that causes diabetes complications.2) In the body, carbonyl groups of reducing sugars react with the amino groups of proteins, lipids, and nucleic acids, irreversibly forming AGEs.3) AGE formation and accumulation occurs gradually with age, even under normal physiological conditions4) and increases with diabetes; AGEs accumulate in an organization. The serum is related to diabetes complications. For example, AGEs accumulate in the skin as a diabetic complication. We have previously reported AGE accumulation in the skin of patients with age-related type 2 diabetes and an associated increase in macrophages.5) These macrophages are involved in inflammation via activating and promoting the production of various cytokines, whose increase with age is associated with chronic inflammation.6)
In addition to macrophages, mast cells, which are also inflammatory cells, are activated by AGEs. Mast cells produce histamine during allergic reactions, which is involved in several physiological and pathological conditions; increased plasma histamine levels have been reported in diabetes. AGEs accumulation, increased mast cell numbers, and associated cytokine production have been observed in the small intestine of patients with diabetes mellitus7); associated chemokine production may also be induced.
The association between diabetes and histamine has previously been assessed, and it has been reported that levels of histamine are elevated in the kidneys, brain, lungs, heart, pancreas, and intestines in individuals with diabetes.8) To date, however, there have been few reports regarding the association between histamine and the small intestine in type 2 diabetes. Furthermore, the effects of diabetes and the maintenance of hyperglycemia with aging on histamine in the small intestine have rarely been investigated.
With regards to the association between diabetes and the gastrointestinal system, gastrointestinal symptoms such as vomiting, constipation, diarrhea, and fecal incontinence have been observed in diabetic patients,9) and in many cases, these symptoms are believed to be associated with abnormal gastrointestinal motility, although the underlying mechanisms have yet to be sufficiently determined.10) In recent years, however, the associations between diseases and the composition of intestinal bacteria have been attracting increasing attention, and it has been reported that abnormalities of the intestinal microbiota are also observed in individuals with type 2 diabetes.11)
Although the mechanisms underlying diabetes and its complications have been studied, age-related changes in the small intestine in diabetes have rarely been investigated. We previously reported AGE accumulation and increased macrophages in diabetes; however, the effect of AGE accumulation on mast cells remains unconfirmed. In this study, the pathological mechanisms of diabetes in the small intestine were investigated with regard to AGEs, mast cell-mediated molecular changes, and cell adhesion molecules using a mouse model of age-related type 2 diabetes.
Specific pathogen-free C57BL/6N and KK-Ay/TaJcl male mice (C57BL/6N background) of different ages (10 and 50 weeks) were obtained from Clare Japan, Inc. (Tokyo, Japan). C57BL/6N mice were used as controls. Mice were individually caged and placed in an air-conditioned room under specific pathogen-free conditions of 23 ± 1 °C and a 12 h light/12 h dark cycle. Food and water were provided ad libitum. The body weight (g) of each mouse was measured weekly. Blood was drawn from the tail vein and blood glucose levels were measured. Animals with blood glucose levels above 300 mg/dL were considered diabetic. The study was approved by the Suzuka University of Medical Sciences Animal Experimentation Ethics Committee on September 25, 2014, and was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals at Suzuka University of Medical Sciences (Approval No: 34). All surgeries were conducted under pentobarbital anesthesia and efforts were made to minimize animal suffering.
Tissue StainingFor histological analysis, the jejunum was collected uniformly 5 cm below the duodenum on the final day of the study period. It was fixed in 4% phosphate-buffered paraformaldehyde and embedded in Tissue-Tek OCT compound (Sakura Finetek Corporation, Tokyo, Japan). Sections of 5 µm thickness were then cut and air-dried at room temperature. Jejunal sections were stained with antibodies for immunofluorescence analysis, as described below.
The following primary antibodies were used: rabbit polyclonal anti-tryptase (1 : 100), rabbit polyclonal anti-DAO (1 : 100; Santa Cruz Biotechnology, CA, U.S.A.), rabbit polyclonal anti-zonula occludens-1 (ZO-1) (1 : 100; Cell Signaling Technology Inc., MA, U.S.A.), rabbit polyclonal anti Claudin1 (1 : 100; Abcam, MA, U.S.A.).
Sections were then incubated with appropriate secondary antibodies (1 : 30 dilution; fluorescein isothiocyanate-conjugated anti-rabbit, anti-mouse, anti-rat, or anti-goat secondary antibodies; Dako Cytomation, Glostrup, Denmark) for 2 h in the dark. Stained samples were observed using fluorescence microscopy. AGE, tryptase, diamine oxidase (DAO), ZO-1, and Claudin1 were measured using ImageJ software ver. 1.53 (National Institutes of Health, MD, U.S.A.) with a constant area, from five random fields of view. These regions are defined as ‘intensity’ in this study.
Analysis of AGEs, Tumor Necrosis Factor (TNF)-α, and HistamineThe jejunum was isolated, homogenized in lysis buffer (Kurabo, Osaka, Japan), and centrifuged at 10000 rpm using Tomy MX-201, after which the supernatant was sampled. AGEs, TNF-α and histamine expression was determined using AGE (Cell Biolabs Inc., CA, U.S.A.), TNF-α (Enzo Life Sciences, NY, U.S.A.) and histamine (Bertin Pharm., Montigny-le-Bretonneux, France) enzyme-linked immunosorbent assay kits as per manufacturer’s instructions. Optical concentrations were measured using a microplate reader (Molecular Devices, CA, U.S.A.).
Statistical AnalysisAll data are expressed as mean ± standard deviation (S.D.). Statistical analyses were performed using Microsoft Excel 2010. One-way ANOVA and Tukey’s post-hoc test were performed using SPSS version 20 (SPSS Inc.). Correlation coefficients were calculated using Pearson's correlation coefficient. Values of p < 0.05 and p < 0.01 were considered statistically significant.
To investigate AGE expression in the small intestine of aging type 2 diabetic mice, we measured the AGE concentration in the small intestine of the KK-Ay/TaJcl group, which was significantly higher in 50-week-old than in 10-week-old mice. In the control group, no differences were found between the 10 and 50-week-old mice. Significantly higher values were observed in KK-Ay/TaJcl mice compared to the control (Fig. 1A). To further confirm that blood glucose levels are elevated in aging type 2 diabetic mice, we measured the levels of blood glucose in KK-Ay/TaJcl mice (Fig. 1B). The findings revealed that compared with mice of 10 weeks of age, KK-Ay/TaJcl group mice were characterized by an increase in blood glucose levels at 50 weeks of age, whereas no significant differences were observed between 10- and 50-week-old control group mice. In addition, relative to the control group, we detected a significant increase in blood glucose levels in the KK-Ay/TaJcl group mice. Furthermore, correlation analysis revealed a positive correlation between blood glucose levels and AGE concentrations in the small intestine (Fig. 1C).
AGEs in the small intestine (A) and blood glucose (B) in age-related type 2 diabetes. Correlation between small intestinal AGEs and blood glucose levels. Values are expressed as mean ± S.D. of five animals. * p < 0.05, ** p < 0.01. N = 5.
Tryptase, a mast cell marker in the small intestine, was also measured. Tryptase activity increased in the small intestine of the KK-Ay/TaJcl group compared to the control group (Figs. 2A, C). In addition, the tryptase levels increased with age in the KK-Ay/TaJcl group, whereas no age-related changes were observed in the control group. The levels of DAO, a histamine-degrading enzyme, in the small intestine were significantly reduced in type 2 diabetic mice compared to that in the controls (Figs. 2B, D). DAO levels further decreased in with increasing age. In the control group, no significant differences were observed between 10 and 50 weeks-old mice.
Tryptase (mast cell marker; A, C) and DAO levels in the small intestine (B, D) in age-related type 2 diabetes. Values are expressed as mean ± S.D. of five animals. * p < 0.05, ** p < 0.01. Scale bar = 100 µm. N = 5.
The levels of the main cell adhesion molecules in the small intestine, ZO-1 and Claudin1, were measured. Both ZO-1 and Claudin1 were reduced in the KK-Ay/TaJcl group compared to the control group (Fig. 3). In addition, the ZO-1 and Claudin1 decreased with age in the KK-Ay/TaJcl group, whereas no age-related changes were observed in the control group.
Values are expressed as mean ± S.D. of five animals. * p < 0.05, ** p < 0.01. Scale bar = 100 µm. N = 5.
Concentrations of the pro-inflammatory cytokines TNF-α and histamine were measured in plasma and small intestine; both TNF-α and histamine were increased in plasma and small intestine in the KK-Ay/TaJcl group compared to controls and further increased with age in the KK-Ay/TaJcl group (Figs. 4A, B). Furthermore, correlation analysis revealed a positive correlation between blood and small intestinal TNF-α concentrations (Fig. 4C).
Plasma and small intestinal TNF-α (A) and histamine (B) concentrations in age-related type 2 diabetes. Correlation between blood TNF-α and small intestinal TNF-α (C). Values are expressed as mean ± S.D. of five animals. * p < 0.05, ** p < 0.01. N = 5.
AGEs increase in the small intestine of aging type 2 diabetic mice. The study shows that type 2 diabetes mellitus increases mast cells and decreases DAO in the small intestine, and that aging further exacerbates these effects. Furthermore, the cell adhesion molecules ZO-1 and Claudin1 in the small intestine are decreased in type 2 diabetes and further reduced with age. Inflammatory cytokines TNF-α and histamine were shown to be increased in both plasma and small intestine in type 2 diabetes and further increased with age.
Mast cells play an important role in allergic reactions and release chemical mediators, including histamines, cytokines, and leukotrienes, when activated.7) AGEs activate mast cells.12) In the present study, we found increased levels of AGEs, mast cells, histamine and TNF-α in the small intestine in type 2 diabetes. The increased AGEs activate mast cells, resulting in increased histamine and TNF-α secretion. Generally, free histamine is degraded by DAO; however, DAO was reduced in the small intestine, suggesting that the degradation of increased histamine was inhibited and maintained instead.
Furthermore, the expression of ZO-1 and Claudin1 in the small intestine decreased. These molecules regulate the permeation of intercellular substances and maintain homeostasis in organisms.13) In type 2 diabetic mice, the reduction of ZO-1 and Claudin1 disrupts adhesion junctions, widening the gap between small intestinal cells, facilitating the entry of various substances into the plasma. This is presumed to result in increased TNF-α and histamine levels in the small intestine being translocated into the plasma.
We indicated that histamine and TNF-α were responsible for the skin deterioration caused by type 2 diabetes.5) Histamine leaks into the blood because of the small intestinal cell adhesion molecule disruption and translocates to the skin, becoming a causal factor. TNF-α is known to be an important aggravating factor in type 2 diabetes.14) We have shown that increased AGEs result in secretion of TNF-α from the skin, liver, and kidneys.15) This study suggests the outflow of TNF-α from the small intestine. However, the mechanisms by which ZO-1 and Claudin-1 levels are reduced in type 2 diabetes remain unknown. Histamine and TNF-α may affect the small intestine in an autocrine manner; however, further studies are required in this regard.
This study contributes to understanding hyperglycemia maintenance caused by age-related type 2 diabetes affecting the small intestine and may help further elucidate the pathological mechanisms of type 2 diabetes and its associated complications for developing novel treatment strategies.
The authors declare no conflict of interest.
