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Approximately 10% of patients with type 2 diabetes suffer from latent autoimmune diabetes in adults (LADA). This study provides a systematic assessment of the pathology of the endocrine pancreas of patients with LADA and for comparison in a first rat model mimicking the characteristics of patients with LADA. Islets in human and rat pancreases were analyzed by immunohistochemistry for immune cell infiltrate composition, by in situ RT-PCR and quantitative real-time PCR of laser microdissected islets for gene expression of proinflammatory cytokines, the proliferation marker proliferating cell nuclear antigen (PCNA), the anti-inflammatory cytokine interleukin (IL) 10, and the apoptosis markers caspase 3 and TUNEL as well as insulin. Human and rat LADA pancreases showed differences in areas of the pancreas with respect to immune cell infiltration and a changed ratio between the number of macrophages and CD8 T cells toward macrophages in the islet infiltrate. Gene expression analyses revealed a changed ratio due to an increase of IL-1β and a decrease of tumor necrosis factor-α. IL-10, PCNA, and insulin expression were increased in the LADA situation, whereas caspase 3 gene expression was reduced. The analyses into the underlying pathology in human as well as rat LADA pancreases provided identical results, allowing the conclusion that LADA is a milder form of autoimmune diabetes in patients of an advanced age. [ABSTRACT FROM AUTHOR]

Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic β cells, it has been proposed that the luminal H2O2 concentration might be very high. As the role of this H2O2 in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2 removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2 significantly with a threefold increase of the EC50 value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with β cell toxic cytokines (IL1β alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2 reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2 toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2 in the ER in spite of a reduced insulin transcription and processing. [ABSTRACT FROM AUTHOR]

The LEW.1 AR1- iddm rat is an animal model of human type 1 diabetes ( T1 D), which arose through a spontaneous mutation within the major histocompatibility complex ( MHC)-congenic background strain LEW.1 AR1. The LEW.1 AR1- iddm rat is characterized by two phenotypes: diabetes development with a diabetes incidence of 60% and a variable T cell frequency in peripheral blood. In this study the immune cell repertoire of LEW.1 AR1- iddm rats was analysed over time from days 30 to 90 of life and compared to the background strain LEW.1 AR1 and the LEW rat strain as well as the LEW.1 WR1 rat strain. The LEW.1 AR1- iddm rats are characterized by a high variability of CD3+, CD4+ and CD8+ T cell frequencies in peripheral blood over time, and the frequency is unique for each animal. The variability within the frequencies resulted in changes of the CD4+ : CD8+ T cell ratio. The other three rat strains studied were characterized by a stable but nevertheless strain-specific T cell frequency resulting in a specific CD4+ : CD8+ T cell ratio. The frequency of natural killer ( NK) cells and B cells in LEW.1 AR1- iddm rats was increased, with a higher variability compared to the other strains. Only monocytes showed no differences in frequency and variability between all strains studied. These variabilities of immune cell frequencies in the LEW.1 AR1- iddm rats might lead to imbalances between autoreactive and regulatory T cells in peripheral blood as a prerequisite for diabetes development. [ABSTRACT FROM AUTHOR]

Progesterone has been associated with the development of gestational diabetes (GD) due to the enhancement of insulin resistance. As β-cell apoptosis participates in type 1 and type 2 diabetes pathophysiology, we proposed the hypothesis that progesterone might contribute to the development of GD through a mechanism that also involves b-cell death. To address this question, RINm5F insulin-producing cells were incubated with progesterone (25-100 μM), in the presence or absence of a-tocopherol (40 μM). After 24 or 48 h, membrane integrity andDNA fragmentation were analyzed by flow cytometry. Caspase activity was used to identify the mode of cell death. The involvement of endoplasmic reticulum stress in the action of progesterone was investigated by western blotting. Oxidative stress was measured by 2',7'-dichlorofluorescein diacetate (DCFDA) oxidation. Isolated rat islets were used in similar experiments in order to confirm the effect of progesterone in primary β-cells. Incubation of RINm5F cells with progesterone increased the number of cells with loss of membrane integrity and DNA fragmentation. Progesterone induced generation of reactive species. Pre-incubation with a-tocopherol attenuated progesterone-induced apoptosis. Western blot analyses revealed increased expression of CREB2 and CHOP in progesteronetreated cells. Progesterone caused apoptotic death of rat islet cells and enhanced generation of reactive species. Our results show that progesterone can be toxic to pancreatic b-cells through an oxidative-stress-dependent mechanism that induces apoptosis. This effect may contribute to the development of GD during pregnancy, particularly under conditions that require administration of pharmacological doses of this hormone. [ABSTRACT FROM AUTHOR]

Previous studies have shown that homocysteine (HC) has a detrimental impact on insulin secretion and pancreatic beta cell function. The aim of the present study was to determine the role of reactive oxygen species (ROS) in the in vitro toxic effects of HC on insulin secretion and function of BRIN-BD11 insulin-secreting cells. In this study, insulin secretion from BRIN-BD11 cellswas determined radioimmunologically, cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and glucokinase activity by a glucose phosphorylation assay following culture with HC plus alloxan (Alx). Treatment with HC resulted in concentrationdependent inhibition of insulin secretion induced by glucose and other insulinotropic agents. HC in combination with Alx resulted in a more pronounced decline in insulin secretion, including that induced by 20 mMalanine, by 43% (P<0.001) and 30 mM KCl by 60% (P<0.001), compared with control culture. The glucokinase phosphorylating capacity in cells cultured with HC plus Alx was significantly lower, compared with control cells. The cells also displayed a significant 84% (P<0.001) decline in cell viability. Prolonged, 72-h culture of insulin-secreting cells with HC followed by 18-h culture without HC did not result in full restoration of beta cell responses to insulinotropic agents. In vitro oxygen consumption was enhanced by a combination of Alx with HC. The study arrived at the conclusion that HC generates ROS in a redox-cycling reaction with Alx that explains the decline in viability of insulin-secreting cells, leading to reduced glucokinase phosphorylating ability, diminished insulin secretory responsiveness and cell death. [ABSTRACT FROM AUTHOR]

The LEW.1AR1- iddm rat is an animal model of spontaneous type 1 diabetes mellitus. This study analysed how adoptive transfer of selective T cell subpopulations affects the incidence of diabetes. CD4+ or CD8+ T cells were isolated from diabetic LEW.1AR1- iddm rats or diabetes-resistant LEW.1AR1 rats. Cells were selectively transferred into athymic LEW.1AR1- Whn rnu or prediabetic LEW.1AR1- iddm rats. The animals were monitored for blood glucose, islet infiltration and immune cell composition of pancreas-draining lymph nodes. After adoptive transfer of CD4+ T cells from diabetic LEW.1AR1- iddm rats into athymic LEW.1AR1- Whn rnu rats, 50% of the recipients developed diabetes. Transfer of CD8+ T cells failed to induce diabetes. Only 10% of the athymic recipients became diabetic after co-transfer of CD4+ and CD8+ T cells. Adoptive transfer of CD8+ T cells from LEW.1AR1 or diabetic LEW.1AR1- iddm rats into prediabetic LEW.1AR1- iddm rats significantly reduced the incidence of diabetes. In protected normoglycaemic animals regulatory CD8+/CD25+ and CD4+/CD25+ T cell subpopulations that were also FOXP3-positive accumulated in the pancreas-draining lymph nodes. In this lymphatic organ, gene expression of anti-inflammatory cytokines was significantly higher than in diabetic rats. Our results show that adoptive transfer of CD4+ but not CD8+ T cells from diabetic LEW.1AR1- iddm rats induced diabetes development. Importantly, CD8+ T cells from diabetic LEW.1AR1- iddm rats and diabetes-resistant LEW.1AR1 rats provided protection against beta cell destruction. The accumulation of regulatory T cells in the pancreas-draining lymph nodes from protected rats indicates that transferred CD8+ T cells may have beneficial effects in the control of beta cell autoimmunity. [ABSTRACT FROM AUTHOR]