IDDM results from a progressive loss of pancreatic beta-cells that, in humans, may be triggered by a combination of genetic and environmental factors. Recently, attention has been focused on the hypothesis that the loss of beta-cells is initiated by inappropriate induction of apoptosis. We now demonstrate that human islets of Langerhans undergo apoptosis upon exposure to interleukin-1beta. The cytokine also sharply increases the number of cells that enter apoptosis on treatment with a stimulatory anti-Fas antibody. Western blotting and immunocytochemistry clearly show for the first time that human pancreatic beta-cells normally express Fas ligand. The results suggest that human islet cells are primed to undergo apoptosis by interleukin-1beta and that this involves the close association between cell-surface Fas and its ligand.

Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B-cell-directed cytotoxicity of cytokines in insulin-dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B-cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B-celliine HIT-TI5 in a dose-and time-dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8-bromo cGMP (100 ~) and dibutyryl cGMP (300 ~) were able to fully relieve this inhibition. In addition, incubation of HIT -T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase (PKG), abolished the induction of cell death in fiT cells in response to either GSNO or cGMP analogues. This effect was dose-dependent over the concentration range of 10--250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT-TI5 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.

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We have shown that nitric oxide treatment for 30-90 min causes inhibition of insulin secretion, DNA damage and disturbs sub-cellular organization in rat and human islets of Langerhans and HIT-T15 cells. Here rat islets and beta-cell lines were treated with various free radical generating systems S-nitrosoglutathione (nitric oxide), xanthine oxidase plus hypoxanthine (reactive oxygen species), 3-morpholinosydnonimine (nitric oxide, super-oxide, peroxynitrite, hydrogen peroxide) and peroxynitrite and their effects over 4 h to 3 days compared with those of the cytokine combination interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma. End points examined were de novo protein synthesis, cellular reducing capacity, morphological changes and apoptosis by acridine orange cytochemistry, DNA gel electrophoresis and electron microscopy. Treatment (24-72 h) with nitric oxide, superoxide, peroxynitrite or combined cytokines differentially decreased redox function and inhibited protein synthesis in rat islets of Langerhans and in insulin-containing cell lines; cytokine effects were arginine and nitric oxide dependent. Peroxynitrite gave rare apoptosis in HIT-T15 cells and superoxide gave none in any cell type, but caused the most beta cell-specific damage in islets. S-nitroso-glutathione was the most effective agent at causing DNA laddering or chromatin margination characteristic of apoptotic cell death in insulin-containing cells. Cytokine-induced apoptosis was observed specifically in islet beta cells, combined cytokine effects on islet function and death most resembled those of the mixed radical donor SIN-1.

The generation of an autoimmune response against islet beta‐cells is central to the pathogenesis of type 1 diabetes mellitus, and this response is driven by the stimulation of autoreactive lymphocytes by components of the beta‐cells themselves. Reactive oxygen species (ROS) have been implicated in the beta‐cell destruction which leads to type 1 diabetes and may modify beta‐cell components so as to enhance their immunogenicity. We investigated the effects of oxidation reactions catalysed by copper or iron on the major beta‐cell autoantigen glutamic acid decarboxylase (GAD). Lysates of purified rat islets were exposed to copper or iron sulphate with or without hydrogen peroxide or ascorbic acid. Immunostaining showed that these treatments generated high molecular weight covalently linked aggregates containing GAD. These are not formed by intermolecular disulphide bonds between cysteine residues since they cannot be resolved into monomeric form when electrophoresed under extreme reducing conditions. There was no modification of insulin or pro‐insulin by ROS. The same oxidative changes to GAD could be induced in viable islet cells treated with copper sulphate and hydrogen peroxide, and thus the modifications are not an artefact of the catalysed oxidation of cell‐free lysates. Sera from patients with type 1 diabetes and stiffman syndrome containing GAD antibodies reacted predominantly with the highest molecular weight modified protein band of GAD: normal human sera did not precipitate GAD. Thus, oxidatively modified aggregates of GAD react with serum antibodies of type 1 diabetes patients and some SMS patients: this is consistent with oxidative modifications of autoantigens being relevant to the pathogenesis of type 1 diabetes.

efaroxan and RX821002, which are related in structure to idazoxan, were much less effective as displacers. 4 In insulin secretion experiments, the ATP-sensitive potassium channel agonist diazoxide (250 ftM) was able to suppress the rise in insulin secretion induced by 20 mM glucose. Both efaroxan and RX821002 (100 ELM) antagonized the inhibitory effect of diazoxide on glucose-induced insulin secretion. By contrast, neither idazoxan (100 M) nor UK14304 (50 gM), was able to overcome significantly the inhibitory effect of diazoxide. 5 The ability of 100 m efaroxan to antagonize the suppression of insulin secretion mediated by diazoxide, was not prevented by idazoxan (up to 100 M) or by UK14304 (up to 50 pM). 6 The results indicate that the stimulatory effects of imidazoline compounds on insulin secretion are not due to interaction with NAIBS similar to those present in rat liver.

The loss of beta-cell mass consequential to the activation of pro-apoptotic signalling events is increasingly recognised as a causal and committed stage in the development of autoimmune, type 1, diabetes mellitus (DM). While the mechanisms responsible for targeted beta-cell loss are multifaceted and difficult to define at a prediabetic stage, there is a need, from a therapeutic perspective, to understand the molecular mechanisms involved. Over recent years the use of animal and cell-line models of DM, together with investigations in isolated ex vivo human islets, have greatly increased our understanding of the processes involved in the pathogenesis of type 1 DM. From this work, several biochemical pathways have emerged that may have future potential for therapeutic intervention. This review looks at the current opinions on the role of apoptosis in beta-cell loss at the molecular level, focusing on a central mechanism for oxidative and nitrosative stress, and suggests biochemical pathways that may have future potential for therapeutic intervention.

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