apyrase and Diabetes-Mellitus--Type-2

Diabetes mellitus encompasses two distinct disease processes: autoimmune Type 1 (T1D) and nonimmune Type 2 (T2D) diabetes. Despite the disparate aetiologies, the disease phenotype of hyperglycemia and the associated complications are similar. In this paper, we discuss the role of the CD39-adenosinergic axis in the pathogenesis of both T1D and T2D, with particular emphasis on the role of CD39 and CD73.

Topics: Adenosine; Animals; Antigens, CD; Apyrase; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Humans; Pancreas; Signal Transduction

Type 1 diabetes (T1D) is an autoimmune disease resulting from an attack by autoreactive T lymphocytes against pancreatic islet β- cells. In recent studies, regulatory T cells (Tregs) have been implicated in the process of T1D. Furthermore, cluster of differentiation 39 (CD39), which is involved in the suppression of inflammation, has been shown to be expressed on Tregs. However, the pathological importance of CD39 to the memory Treg population remains unclear.. This study investigated Treg subsets, focusing on resting, effector, and memory Tregs, and determined CD39 expression on Tregs. In addition, changes in Treg subsets and Treg-associated cytokine secretion after CD3/CD28 stimulation of peripheral blood mononuclear cells were evaluated in diabetic patients and healthy controls. The suppressive function of Tregs was measured using the mixed lymphocyte reaction (MLR) test.. There was a higher percentage of memory Tregs in T1D patients than healthy controls. However, Tregs in T1D patients showed impaired suppression, with low forkhead box P3 (Foxp3) expression and low serum interleukin (IL)-10 levels. Furthermore, CD39 expression on Tregs, and on memory Tregs in particular, was lower in T1D patients than healthy controls. After stimulation, the percentage of resting Tregs was decreased and that of effector/memory Tregs was increased in both healthy controls and T1D patients, but CD39 expression on effector/memory Tregs was still lower and there was no increase in IL-10 secretion in T1D patients.. The defective suppressive function of Tregs in T1D patients is due to lower expression of CD39 on memory Tregs.. 摘要: 背景 1型糖尿病是一种由自身T淋巴细胞攻击胰岛β细胞而造成的自身免疫性疾病。最近有研究表明调节性T细胞(regulatory T cells，Tregs)在1型糖尿病发病过程中发挥作用，此外也有研究证明分化抗原簇39(CD39)参与炎症抑制过程，且在Tregs上表达。然而，CD39在记忆型Treg亚群中病理学上的重要性仍未阐明。 方法 该研究将分析Tregs亚群，包括静息、效应和记忆Tregs亚群，检测CD39是否在这些Tregs亚群中表达。在患者和健康对照者中，以CD3/CD28刺激外周血单核细胞，观察Tregs亚群及Treg相关细胞因子分泌情况的变化。同时，在混合淋巴细胞反应中检测患者和健康对照Treg的抑制功能。 结果 虽然该研究发现1型糖尿病患者中记忆型Treg亚群比例高于健康对照，但是，1型糖尿病患者Treg的抑制功能受损，同时其Foxp3的表达降低及血清中IL-10分泌水平降低，并且CD39在Tregs中的表达，尤其是在记忆Tregs中的表达低于健康对照。在CD3/CD28磁珠刺激后，不管是患者还是健康对照，静息Treg亚群的比例降低，效应/记忆Treg亚群升高。但是1型糖尿病患者中，PBMCs经过刺激后，CD39在效应/记忆Treg亚群中仍然维持低表达，而IL-10的分泌也并没有上升。 结论 1型糖尿病患者中Treg抑制功能的受损是由于CD39在记忆Treg亚群中的表达降低所致。.

Topics: Adult; Apyrase; Biomarkers; Blood Glucose; Case-Control Studies; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Follow-Up Studies; Forkhead Transcription Factors; Glycated Hemoglobin; Humans; Immunologic Memory; Interleukin-10; Leukocytes, Mononuclear; Male; Middle Aged; Prognosis; T-Lymphocytes, Regulatory

We investigated the relationship between inflammation, neuronal loss, and expression of indoleamine 2, 3-dioxygenase (IDO) and quinolinic acid (QUIN) in the retina of subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) and in the retina of rats with T1D.. Retinas from T1D (n = 7), T2D (n = 13), and 20 age-matched nondiabetic human donors and from T1D (n = 3) and control rats (n = 3) were examined using immunohistochemistry for IDO, QUIN, cluster of differentiation 39 (CD39), ionized calcium-binding adaptor molecule (Iba-1, for macrophages and microglia), Vimentin (VIM; for Müller cells), neuronal nuclei (NeuN; for neurons), and UEA1 lectin (for blood vessels).. Based on morphologic criteria, CD39+/ionized calcium binding adaptor molecule 1(Iba-1+) resident microglia and CD39-/Iba-1+ bone marrow-derived macrophages were present at higher density in T1D (13% increase) and T2D (26% increase) human retinas when compared with controls. The density and brightness of IDO+ microglia were increased in both T1D and T2D human retinas. The intensity of QUIN+ expression on CD39+ microglia and VIM+ Müller cells was greatly increased in both human T1D and T2D retinas. T1D retinas showed a 63% loss of NeuN+ neurons and T2D retinas lost approximately 43% when compared with nondiabetic human retinas. Few QUIN+ microglia-like cells were seen in nondiabetic retinas, but the numbers increased 18-fold in T1D and 7-fold in T2D in the central retina. In T1D rat retinas, the density of IDO+ microglia increased 2.8-fold and brightness increased 2.1-fold when compared with controls.. Our findings suggest that IDO and QUIN expression in the retinas of diabetic rats and humans could contribute to the neuronal degeneration that is characteristic of diabetic retinopathy.

Topics: Aged; Animals; Antigens, CD; Antigens, Nuclear; Apyrase; Biomarkers; Calcium-Binding Proteins; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; DNA-Binding Proteins; Ependymoglial Cells; Female; Fluorescent Antibody Technique, Indirect; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Male; Microfilament Proteins; Microglia; Microscopy, Confocal; Middle Aged; Nerve Tissue Proteins; Quinolinic Acid; Rats; Rats, Sprague-Dawley; Retina; Vimentin

Regulatory T cells have various mechanisms to suppress the inflammatory response, among these, the modulation of the microenvironment through adenosine and with the participation of CD39, CD73 and A2A. The aim of this study was to assess the expression of CD73 and A2A in immune cells and the effect of activation of A2A by an adenosine analogue on apoptosis in patients with obesity and type 2 diabetes mellitus (T2D). CD73 and A2A expression were analyzed by flow cytometry in lymphocyte subpopulations from patients with obesity (n = 22), T2D (n = 22), and healthy subjects (n = 20). Lymphocytes were treated with the selective A2A antagonist (ZM241385) or the selective A2A agonist (CGS21680), and apoptotic cells were detected by Annexin V. We found an increased expression of CD39 coupled to a decrease in CD73 in the patient groups with obesity and T2D compared to the control group in the different studied lymphocyte subpopulations. A2A expression was found to be increased in different subpopulations of lymphocytes from T2D patients. We also detected positive correlations between CD39+ cells and age and BMI. Meanwhile, CD73+ cells showed negative correlations with age, WHR, BMI, FPG, HbAc1, triglycerides and cholesterol. Moreover, an increase in the percentage of apoptotic cells from T2D patients with regard to the groups with obesity and control was observed. In addition, the CD8+ T cells of patients with T2D exhibited decreased apoptosis when treated with the A2A agonist. In conclusion, our data suggest a possible role for CD73 and A2A in inflammation observed in patients with T2D and obesity mediated via apoptosis.

Topics: 5'-Nucleotidase; Adenosine; Adult; Antigens, CD; Apoptosis; Apyrase; Body Mass Index; CD8-Positive T-Lymphocytes; Diabetes Mellitus, Type 2; Gene Expression Regulation; GPI-Linked Proteins; Humans; Inflammation; Lymphocyte Subsets; Lymphocytes; Obesity; Phenethylamines; Receptor, Adenosine A2A; Triazines; Triazoles

Th17 cells are involved in the pathogenesis of multiple inflammatory diseases such as type two diabetes (T2D). CD39(+) Treg cells have been implicated as responsible for suppressing Th17 cells. The aim of this study was to evaluate the number and function of CD4(+)CD25(high)CD39(+) Treg and Th17 cells in peripheral blood mononuclear cells (PBMC) from T2D patients and healthy control subjects. The Th17 cells were detected in PBMC under culture with human anti-CD3/CD28 and PMA/ionomycin and the levels of IL-17 were assessed by ELISA and qPCR. The T2D patients with obesity showed significantly lower percentages of CD39(+) Treg cells. A negative correlation between CD39(+) Treg cells and weight, and body mass index was detected. In contrast, the low levels of CD4(+)IL-17(+) cells in overweight and obese T2D patients showed a positive correlation with glucose and HbA1c. Additionally, we found a subpopulation of Th17 cells that express CD39 and were correlated with glucose and HbA1c. Our findings suggest that the expression of CD39 on Treg cells and also in CD4(+)IL-17(+) cells from T2D patients is related to hyperglycemia as well as to overweight and obesity and therefore may participate as a modulator of the effector capacity of Th17 cells.

Topics: Adult; Aged; Antigens, CD; Apyrase; Biomarkers; Case-Control Studies; Cytokines; Diabetes Mellitus, Type 2; Female; Forkhead Transcription Factors; Gene Expression; Humans; Immunophenotyping; Lymphocyte Count; Male; Middle Aged; Phenotype; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Th17 Cells

Chronic inflammation is an important contributor to the insulin resistance observed in type 2 diabetes (T2D). We evaluated the expression and function of the P2X(7) receptor and CD39/Entpd1, molecules involved in the cellular regulation of inflammation, in peripheral blood mononuclear cells from T2D patients, and their correlation with the concentration of HbA1c in blood. T2D patients with deficient metabolic control (DC) showed increased proportion of P2X(7)(+) cells compared with healthy individuals; T2D-DC subjects also displayed higher proportion of CD14(+), CD4(+) and CD19(+) subpopulations of P2X(7)(+) cells when compared with T2D patients with acceptable metabolic control. A significant association was observed between the proportion of P2X(7)(+)CD14(+) cells and blood concentration of LDL-c. In addition, the percentages of CD39(+) cells and CD39(+)CD19(+) cells were significantly associated with HbA1c and fasting plasma glucose levels. No changes were observed in the function of P2X(7)(+) cells from T2D patients; however, enhanced CD39/Entpd1 enzyme activity and low serum levels of IL-17 were detected. Therefore, CD39(+) cells could have a balancing regulatory role in the inflammatory process observed in patients with T2D.

Topics: Adenosine Triphosphate; Adult; Antigens, CD; Apyrase; Blood Glucose; Cholesterol, LDL; Diabetes Mellitus, Type 2; Female; Glycated Hemoglobin; Humans; Interleukin-17; Interleukin-1beta; L-Selectin; Leukocytes, Mononuclear; Lipopolysaccharides; Male; Middle Aged; Receptors, Purinergic P2X4; Receptors, Purinergic P2X7; Young Adult

The vascular ectonucleotidase ENTPD1 protects against renal injury and modulates glucose homeostasis in mouse models. We sought to determine whether human variation in ENTPD1 influences predisposition to diabetes or diabetic nephropathy.. We analyzed ENTPD1 single nucleotide polymorphisms (SNPs) in 363 African American control subjects, 380 subjects with type 2 diabetes and end-stage renal disease (DM-ESRD), and 326 subjects with ESRD unrelated to diabetes (non-DM-ESRD). Using human cell lines, we correlated disease-associated ENTPD1 haplotypes with ENTPD1 gene expression. Finally, we studied consequences of ENTPD1 deletion in a mouse model of type 2 diabetes (db/db).. A common ENTPD1 two-SNP haplotype was associated with increased risk for DM-ESRD (P = 0.0027), and an uncommon four-SNP haplotype was associated with protection against DM-ESRD (P = 0.004). These haplotypes correlated with ENTPD1 gene expression levels in human cell lines in vitro. Subjects with high ENTPD1-expressing haplotypes were enriched in the DM-ESRD group. By crossing ENTPD1-null mice with db mice, we show that ENTPD1 deletion has prominent effects on metabolic syndrome traits. Specifically, deletion of ENTPD1 lowered glucose levels in control (db/-) mice with one functional leptin receptor and dramatically lowered weights in db/db mice with no functional leptin receptors. Similar effects were seen in aged ENTPD1-null mice with normal leptin receptors.. ENTPD1 polymorphisms appear to influence susceptibility to type 2 diabetes and/or diabetic nephropathy in African Americans. Studies in human cell lines and in vivo mouse data support a potential role for ENTPD1 genetic variation in susceptibility to type 2 diabetes.

Topics: Animals; Antigens, CD; Apyrase; Black People; Cell Line; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Humans; Kidney Failure, Chronic; Mice; Mice, Knockout; Polymorphism, Single Nucleotide; Sequence Deletion

We have investigated expression and function of the P2X7 receptor in fibroblasts from healthy subjects and patients with type 2 diabetes.. Fibroblasts were isolated from skin biopsies. P2X7 receptor expression in both cell populations was measured by functional assays, RT-PCR, fluorescence-activated cell sorter, and immunoblotting. We found that fibroblasts from diabetic subjects are characterized by enhanced P2X7-mediated responses as indicated by increased shape changes, microvesiculation, enhanced fibronectin and interleukin 6 secretion, and accelerated apoptosis. These responses were blocked by preincubation with the P2X blockers KN-62, oxidized ATP, or pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid). Furthermore, we also found a higher level of spontaneous fibronectin secretion and of apoptosis in fibroblasts from diabetic compared with healthy subjects. Both higher basal level of fibronectin secretion and spontaneous rate of apoptosis were likely attributable to the increased pericellular concentration of ATP because fibroblasts from diabetic subjects released 3x as much ATP into the supernatants compared with fibroblasts from healthy subjects.. We conclude that fibroblasts from type 2 diabetes patients are characterized by a hyperactive purinergic loop based either on a higher level of ATP release or on increased P2X7 reactivity.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Diphosphate; Adenosine Triphosphate; Apoptosis; Apyrase; Autocrine Communication; Cell Shape; Cytidine Triphosphate; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Interleukin-6; Membrane Potentials; Paracrine Communication; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Uridine Diphosphate; Uridine Triphosphate

Extracellular ATP is an ubiquitous mediator that regulates several cellular functions via specific P2 plasma membrane receptors (P2Rs), for which a role in modulating intracellular glucose metabolism has been recently suggested. We have investigated glucose uptake in response to P2Rs stimulation in fibroblasts from type 2 diabetic (T2D) patients and control subjects. P2Rs expression was evaluated by RT-PCR; intracellular calcium release by fluorometry; glucose transporter (GLUT1) translocation by immunoblotting and chemiluminescence; glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose (2-DOG) and ATP by luminometry. Cells from T2D patients, in contrast to those from healthy controls, showed no increase in glucose uptake after ATP stimulation; extracellular ATP caused, however, a similar GLUT1 recruitment to the plasma membrane in both groups. P2Rs expression did not differ between fibroblasts from diabetic and healthy subjects, but while plasma membrane depolarization, a P2X-mediated response was similar in both groups, no evident intracellular calcium increase was detectable in the cells from the former group. The calcium response in fibroblasts from diabetics was restored by co-incubation with apyrase or hexokinase, suggesting that P2YRs in those cells were normally expressed but chronically desensitised. In support to this finding, fibroblasts from T2D subjects secreted a two-fold larger amount of ATP compared to controls. Pre-treatment with apyrase or hexokinase also restored ATP stimulated glucose uptake in fibroblasts from diabetic subjects. These results suggest that extracellular ATP plays a role in the modulation of glucose transport via GLUT1, and that the P2Y-dependent GLUT1 activation is deficient in fibroblasts from T2D individuals. Our observations may point to additional therapeutic targets for improving glucose utilization in diabetes.

Topics: Adenosine Triphosphate; Apyrase; Biological Transport; Calcium; Calcium Signaling; Cell Membrane; Cells, Cultured; Diabetes Mellitus, Type 2; Energy Metabolism; Extracellular Fluid; Fibroblasts; Glucose; Glucose Transporter Type 1; Hexokinase; Humans; Insulin Resistance; Membrane Potentials; Monosaccharide Transport Proteins; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; RNA, Messenger

The authors used histochemical analysis to determine whether increased vascular endothelial growth factor (VEGF) immunoreactivity in diabetic retinal vessels is related to increased vascular permeability, as indicated by human serum albumin (HSA) immunostaining, or to presumed retinal hypoxia as demonstrated by decreased vascularity. A correlation between VEGF and HSA in cryosections with angiopathic changes in the adenosine diphosphatase (ADPase) flat-embedded fellow retinas was sought. Because VEGF is a heparin-binding protein, the relation between VEGF and heparan sulfate proteoglycan (HSPG) immunoreactivities was also investigated.. Cryopreserved eyes from 18 diabetic and 9 nondiabetic subjects removed after death were sectioned and immunohistochemical analysis was performed with antibodies against VEGF, HSA, HSPG, vWf (von Willebrand factor), and collagen IV. The fellow retinas were prepared by our ADPase flat-embedding technique to determine the degree of diabetic retinopathy. The number of positive vessels for each antibody and antibody localizations were determined by light microscopy.. The average number of VEGF-stained vessels in diabetic retinas was significantly higher than in nondiabetic retinas (P = 0.04). In diabetic retinas, there was a positive correlation between the distribution of VEGF-positive vessels and the distribution of HSA- and HSPG-positive vessels. No such correlation was observed in nondiabetic eyes. In many cases, HSPG immunoreactivity appeared colocalized with VEGF immunoreactivity, suggesting VEGF binding to HSPG. The comparison with the ADPase flat-embedded fellow retinas suggested that increased VEGF immunoreactivity and vascular permeability may occur before morphologic changes in the vasculature.. Vascular endothelial growth factor immunoreactivity was correlated with increased vascular permeability to macromolecules and appears to be increased in diabetic subjects before the onset of retinopathy.

Topics: Adult; Aged; Aged, 80 and over; Apyrase; Capillary Permeability; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelial Growth Factors; Heparan Sulfate Proteoglycans; Humans; Immunoenzyme Techniques; Lymphokines; Middle Aged; Retinal Vessels; Serum Albumin; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors