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Abstract: Type 1 diabetes results from destruction of pancreatic β cells by β cell-specific autoreactive T cells in the nonobese diabetic (NOD) mouse. Defects in thymic negative selection are thought to result in failure to delete potential β cell-reactive T cells, contributing to the development of autoimmune diabetes. We investigated this possibility by comparing the deletion profile of double-positive (DP) thymocytes in NOD mice with diabetes-resistant strains of mice after anti-CD3 Ab treatment to trigger the TCR-mediated signaling pathway. We found that immature NOD CD4+CD8+ DP thymocytes have a lower activation threshold than C57BL/6 and Balb/c thymocytes. This was confirmed by showing that NOD DP thymocytes have a higher level of ERK and JNK phosphorylation. The low activation threshold of immature thymocytes resulted in rapid deletion of strongly activated immature DP thymocytes by negative selection, whereas weakly activated immature thymocytes differentiated more efficiently into CD69+CD3high DP thymocytes by positive selection. SP thymocytes, particularly CD4−CD8+ T cells that were efficiently generated from activated DP thymocytes, could induce severe insulitis and diabetes in NOD.scid mice. We conclude that the development of autoreactive diabetogenic T cells results from inordinate positive selection due to the low activation threshold of DP thymocytes in NOD mice. [Copyright &y& Elsevier]

Inflammation markers in zebrafish embryos reflect a toxic response that is common to other animal models and humans. Free fatty acids (FFAs) are known to cause damage in various tissues by inducing inflammation. In this study, we investigated whether a FFA (palmitate) induces inflammation in zebrafish embryos. Nitrous oxide (NO) production and cyclooxygenase-2 (COX-2) mRNA expression were increased in palmitate-treated zebrafish embryos in a dose-dependent manner. mRNA expression of pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α), were also increased. Additionally, the mRNA expression of p65 nuclear factor-kB and I-kB-α were significantly increased after palmitate-treatment. Increased reactive oxygen species (ROS) expression was observed in palmitate-treated zebrafish embryos as well as pericardial edema. Additionally, mRNA expression of pro-inflammatory cytokines were increased in zebrafish liver and pancreas fed with palmitate-contained diet. Taken together, these results indicated that palmitate increases pro-inflammatory mediators in zebrafish embryos, suggesting that zebrafish could be an alternative animal model for inflammatory disease including diabetes. [ABSTRACT FROM AUTHOR]

Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H 2 O 2 . Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factor erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element ( ARE ), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study is to investigate the effect of mitochondrial metabolism on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death. Long-term treatment of INS-1 cells with HG/PA impaired energy-producing metabolism accompanying with depletion of TCA cycle intermediates. Whereas an inhibitor of carnitine palmitoyl transferase 1 augmented HG/PA-induced INS-1 cell death, stimulators of fatty acid oxidation protected the cells against the HG/PA-induced death. Furthermore, whereas mitochondrial pyruvate carboxylase inhibitor phenylacetic acid augmented HG/PA-induced INS-1 cell death, supplementation of TCA cycle metabolites including leucine/glutamine, methyl succinate/α-ketoisocaproic acid, dimethyl malate, and valeric acid or treatment with a glutamate dehydrogenase activator, aminobicyclo-heptane-2-carboxylic acid (BCH), significantly protected the cells against the HG/PA-induced death. In particular, the mitochondrial tricarboxylate carrier inhibitor, benzene tricarboxylate (BTA), also showed a strong protective effect on the HG/PA-induced INS-1 cell death. Knockdown of glutamate dehydrogenase or tricarboxylate carrier augmented or reduced the HG/PA-induced INS-1 cell death, respectively. Both BCH and BTA restored HG/PA-induced reduction of energy metabolism as well as depletion of TCA intermediates. These data suggest that depletion of the TCA cycle intermediate pool and impaired energy-producing metabolism may play a role in HG/PA-induced cytotoxicity to beta cells and thus, HG/PA-induced beta cell glucolipotoxicity can be protected by nutritional or pharmacological maneuver enhancing anaplerosis or reducing cataplerosis. [ABSTRACT FROM AUTHOR]

Abstract: Free fatty acids (FFAs) are believed to be a stimulus to elicit beta cell dysfunction. The present study was undertaken to determine whether endoplasmic reticulum (ER) stress was involved in palmitate-induced inhibition of glucose-stimulated insulin secretion (GSIS) and whether reduction of ER stress using a chemical chaperone restored the GSIS-inhibition. Treatment of INS-1 cells with 300μM palmitate for 24h elicited ER stress, showing increased levels of phospho-eIF2α, Bip and spliced XBP, and also induced GSIS-inhibition without reduction of cell viability. Replenishment with 4-phenyl butyric acid (4-PBA) as a chemical chaperone reduced the palmitate-induced-ER stress and significantly reversed the palmitate-induced GSIS-inhibition. Furthermore, 4-PBA ameliorated palmitate-induced GSIS-inhibition in primary rat islet cells. These data suggested that ER stress was involved in FFA-induced GSIS-inhibition and that the FFA-induced beta cell dysfunction could be ameliorated by treatment with a chemical chaperone. [Copyright &y& Elsevier]

Gamma interferon (IFN-γ) has been thought to play an important role in the pathogenesis of diabetes. This report determines if rIFN-γ administration to NOD mice paradoxically inhibits the development of diabetes. Injections of recombinant rIFN-γ of 5×103, 20×103, and 100×103 units, dose dependently inhibited the development of diabetes. The maximal rIFN-γ dose decreased the incidence of diabetes from 74% in control animals to 42%. 100×103 unit rIFN-γ dose significantly decreased insulitis score, and increased islet number. The development of diabetes in irradiated NOD mice was slower in animals injected with spleen cells from rIFN-γ treated than from saline treated NOD mice suggesting that rIFN-γ decreases anti-islet effector cell activity. The susceptibility to apoptosis was increased in splenic cells of rIFN-γ treated mice. The expressions of the co-stimulatory molecules B7-2 and ICAM-1 were significantly increased in spleen cells of rIFN-γ treated mice while the expression of MHC class I was decreased. In vitro studies demonstrated that NOD mouse mononuclear spleen cells preincubated with rIFN-γ and subsequently cocultured with responder cells, potently inhibited responder T-cell proliferative responses. rIFN-γ administration decreased IL-12 and IL-2 mRNA expression in spleen cells while increasing IL-1 expression. In conclusion, rIFN-γ inhibits the diabetic process in NOD mice by decreasing anti-islet effector activity and in turn decreasing insulitis and islet destruction. The suppression of Th1 cell related cytokines and/or augmentation of the macrophage cytokine IL-1 may play a role in the diabetes sparing effect of rIFN-γ. [Copyright &y& Elsevier]