Anne C. Loweth

Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15

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‐cell line HIT‐T15 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 μM) and dibutyryl cGMP (300 μM) 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 HIT 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‐T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.

Superoxide, nitric oxide, peroxynitrite and cytokine combinations all cause functional impairment and morphological changes in rat islets of Langerhans and insulin secreting cell lines, but dictate cell death by different mechanisms

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-1β, tumour necrosis factor-α and interferon-γ. 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.

Islet glutamic acid decarboxylase modified by reactive oxygen species is recognized by antibodies from patients with type 1 diabetes mellitus

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.

Stimulation of insulin secretion by imidazoline compounds is not due to interaction with non-adrenoceptor idazoxan binding sites

1 The potency of interaction of several imidazoline compounds with non‐adrenoceptor idazoxan binding sites (NAIBS) in rat liver membranes was compared with their ability to alter insulin secretion from rat pancreatic islets. 2 NAIBS could be labelled specifically with [3H]‐idazoxan in both rat liver membranes and in rat islet homogenates. Liver binding sites exhibited a KD for [3H]‐idazoxan of 24 nm and a Bmax of 264 fmol mg−1 protein. 3 Binding of [3H]‐idazoxan to NAIBS in rat liver membranes was displaced effectively by unlabelled idazoxan (IC50 0.1 μm) and by UK14304 (IC50 0.5 μm). However, two other imidazoline compounds 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 μm) was able to suppress the rise in insulin secretion induced by 20 mm glucose. Both efaroxan and RX821002 (100 μm) antagonized the inhibitory effect of diazoxide on glucose‐induced insulin secretion. By contrast, neither idazoxan (100 μm) nor UK14304 (50 μm), was able to overcome significantly the inhibitory effect of diazoxide. 5 The ability of 100 μm efaroxan to antagonize the suppression of insulin secretion mediated by diazoxide, was not prevented by idazoxan (up to 100 μm) or by UK14304 (up to 50 μm). 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.

Phospholipase A2 expression in human and rodent insulin-secreting cells.

The expression of different isoforms of phospholipase A2 in human and rat islets of Langerhans and in the clonal B-cell lines, HIT-T15 and RINm5F has been investigated, using polyclonal antisera specific to human cytosolic (cPLA2) or human Groups I and II secretory (sPLA2) isoforms. Abundant levels of a 100-kDa protein corresponding to cPLA2 were detected in cytosolic extracts of human islets. A 100-kDa cPLA2 was not detectable in rat islets, RINm5F or HIT-T15 cells using an anti-cPLA2 serum raised against cloned human cPLA2 cDNA, despite the antiserum being cross-reactive with cPLA2 from rat kidney. Human and rat islets were found to express a 21-kDa protein immunoreactive with Group I sPLA2 antiserum. Group II sPLA2 was not detected in human or rat islets. RIN cells did not express detectable levels of either Group I or Group II sPLA2, but HIT cells expressed variable quantities of Group II sPLA2. These differences in PLA2 expression suggest that caution should be exercised when extrapolating conclusions about lipid-derived signalling molecules from insulin-secreting cell lines to normal islets of Langerhans.