Irene C. Green

Inhibition of insulin secretion by interleukin-1β and tumour necrosis factor-α via an L-arginine-dependent nitric oxide generating mechanism

Inhibition of glucose‐induced insulin secretion by interleukin‐1β (IL‐1β), or IL‐1β plus tumour necrosis factor‐α (TNF‐α), was less marked when rat islets of Langerhans were cultured for 12 h with these cytokines in L‐arginine‐free medium as opposed to medium containing L‐arginine (1 mM). Inhibition of secretion by IL‐1β was further alleviated when islets were maintained in L‐arginine‐free medium supplemented with N‐ω‐nitro‐L‐arginine methyl ester (NAME), while synergism between IL‐1β plus TNF‐α was completely abolished. Tissue culture medium nitrite levels were raised in islets treated with IL‐1β or TNF‐α (48 h). Cytokine‐stimulated nitrite production was not observed in islets cultured with NAME (1 mM). In conclusion, an L‐arginine‐dependent nitric oxide generating mechanism is involved in the inhibition of insulin secretion by IL‐1β, and accounts for the phenomenon of synergism between IL‐1β and TNF‐α.

Nitric oxide synthesis and action in an invertebrate brain

Nitric oxide (NO) is synthesized in mammalian neurons by Ca2+/calmodulin activated NO synthase and functions as a signalling molecule by activating soluble guanylyl cyclases in target cells. We demonstrate here that both NO synthase and NO-activated guanylyl cyclase are present in the brain of the locust Schistocerca gregaria. Our observations indicate, for the first time, that the NO-cyclic GMP signalling pathway exists in invertebrate nervous systems.

Endogenous nitric oxide induced by interleukin-1β in rat islets of Langerhans and HIT-T15 cells causes significant DNA damage as measured by the ‘comet’ assay

We have used the comet assay (single cell gel electrophoresis) to measure nitric oxide‐induced DNA damage in rat islets of Langerhans and insulin‐containing HIT‐T15 cells. Damage was induced following treatment with the nitric oxide donor SIN‐1, which also releases Superoxide, but was not reduced by exogenous Superoxide dismutase, suggesting that nitric oxide itself, rather than Superoxide or peroxynitrite may be the active species. The DNA damaging effect of nitric oxide was easily detectable at the earliest time point tested (15 min). Damage also resulted following induction of nitric oxide synthase by the cytokine interleukin‐1β in both islets and HIT‐T15 cells and was prevented by replacing the substrate, arginine, with nitromonomethyl arginine. Thus intracellular levels of nitric oxide generated by interleukin‐1β‐induced nitric oxide synthase were sufficient to cause DNA damage in islet cells and HIT‐T15 cells.

Tumor Necrosis Factor-α and Interferon-γ Inhibit Insulin Secretion and Cause DNA Damage in Unweaned-Rat Islets: Extent of Nitric Oxide Involvement

Nitric oxide has been implicated as one possible mediator of interleukin-1β (IL-1)-induced inhibition of insulin secretion and islet cell damage. The aim of this study was to define the effects of tumor necrosis factor-α (TNF) and interferon-γ (IFN) on nitric oxide production, insulin secretion, and DNA damage in islets from unweaned rats. Treatment of islets with 0.5–500 U/ml of either TNF or IFN on their own inhibited glucose-stimulated insulin secretion in a dose-dependent manner (minimum effective dose 5 U/ml). In combination, the cytokines exerted a pronounced synergistic inhibitory effect on secretion and were equipotent at causing a significant and concentration-dependent increase in culture medium nitrite levels, islet cyclic GMP formation, and DNA damage. Used alone or in combination, TNF and IFN significantly enhanced the activity of inducible nitric oxide synthase as determined by measuring the conversion of 14C-labeled arginine to 14C-labeled citrulline and nitric oxide. Use of arginine-free medium, without or with NG-monomethyl-L-arginine, resulted in inhibition of nitrite formation by 5–1,000 U/ml IFN + TNF and partial restoration of the insulin secretory response to glucose. Treatment of rat islets with increasing doses of TNF + IFN (5, 50, and 500 U/ml) resulted in a progressive increase in DNA damage, as shown by the comet assay, which detects DNA strand breaks in individual islet cells. The DNA damage caused by an intermediate concentration (50 U/ml) of TNF + IFN was comparable to that generated by IL-1 when used at 20 U/ml. We conclude that TNF and IFN induce nitric oxide formation, which partially inhibits glucose-induced insulin secretion and causes significant DNA strand breakage, but that as cytokine concentrations increase, non-nitric-oxide-mediated events predominate.

The location of VIP in the pancreas of man and rat

SummaryVIP has powerful stimulatory effects on both endocrine and exocrine pancreas but its localisation within the gland has not been established. In this study, human pancreas was obtained fresh at surgery (eleven) or within four hours of death (seven). The pancreas was also removed from rats (twenty-two). Immunocytochemical staining showed VIP to be present in fine nerve fibres in all areas of the pancreas. Many fibres were seen in the exocrine pancreas, running between the acini, and around ducts and blood vessels. In addition, dense networks of fibres were observed forming meshes around islets and occasional ganglia were found containing immunoreactive cell bodies. In general, there were fewer VIP fibres in the rat pancreas than in the human, but overall distribution was identical. The mean VIP content of whole human pancreatic tissue was 42±10 pmol/g wet weight (38±9 pmol/g in head, 49±6 pmol/g in body and 42±11 pmol/g in tail). Whole rat pancreatic tissue contained 28±7 pmol/g wet weight while preparations of isolated islets were found to contain 374±30 pmol/g. It is possible that the heavy VIP innervation of the islets described here indicates a role in the regulation of islet hormone release.

Nitric oxide donors decrease the function and survival of human pancreatic islets.

Nitric oxide (NO) has been proposed as a possible mediator of beta-cell damage in human IDDM. This hypothesis is based on in vitro studies with rodent pancreatic islets. In the present study we examined whether human beta-cells are affected by NO. In view of species differences in beta-cell sensitivity to damaging agents, rat islets were investigated in parallel. Isolated islets were exposed for 90 min to different concentrations of three chemically unrelated NO donors, SIN-1, GSNO or RBS. At the end of this incubation, human insulin release was mostly similar in control and NO-treated islets but, 48 h later, islet retrieval, islet DNA and insulin content, and glucose-induced insulin release were markedly lower in islets exposed to NO donors. Rat islets were already inhibited during the initial 90 min; 48 h later their loss in beta-cell function was similar to that in human islets. Nicotinamide or succinic acid monomethyl ester partially protected against SIN-1 induced islet cell loss, but not against the functional inhibition of human pancreatic islets. Exposure of human or rat islets to RBS was associated with significant DNA strand breakage, as judged by the comet assay (single cell gel electrophoresis) and by ultrastructural signs of cell damage. DNA damage was more severe in rat islet cells exposed to similar amounts of RBS. It is concluded that NO donors can damage human pancreatic islets, an effect paralleled by induction of nuclear DNA strand breaks.

Effect of enkephalins and morphine on insulin secretion from isolated rat islets.

SummaryThe direct effects of an enkephalin analogue, (D-Ala2/MePhe4/Met/(O)-ol) enkephalin (DAMME), on insulin release from isolated islets of Langerhans of the rat have been investigated. DAMME had a dose-dependent effect on insulin secretion: low concentrations (10−10 to 10−8 mol/l) were stimulatory while high concentrations (10−5mol/l) were inhibitory in the presence of 8 mmol/l glucose. Similar effects were found with met-enkephalin, and with the longer acting alanine substituted metenkephalin. Morphine sulphate (5 sx 10−7 mol/l) also stimulated insulin release. The effects of enkephalin and morphine were blocked by the specific opiate antagonist naloxone hydrochloride (1.2 × 10−6 mol/l). The insulin secretory response of perifused islets to enkephalins and morphine was rapid, corresponding to the first phase of glucose induced insulin release. These observations suggest that there may be opiate receptors in islets, and that opioid peptides could modulate insulin release.

Fatty acid protection from palmitic acid-induced apoptosis is lost following PI3-kinase inhibition

We have previously shown that saturated fatty acids induce DNA damage and cause apoptotic cell death in insulin-producing β-cells. Here we examine further the effects of single or combined dietary fatty acids on RINm5F survival or cell death signalling. Palmitate and stearate, but not linoleate, oleate or palmitoylmethyl ester, induced growth inhibition and increased apoptosis in RINm5F cells following 24 h exposure. Co-incubation with inhibitors of ceramide synthesis, myriocin or fumonisin B1, did not improve viability of palmitic acid treated RINm5F cells. The inhibitor of inducible nitric oxide synthase, 1400 W, similarly had no protective effect. However, linoleic acid protected against palmitic acid-induced apoptotic and necrotic cell death. The specific pharmacological inhibitors of phosphatidylinositol 3-kinase, LY294002 and wortmannin, abolished the protective effect of linoleic acid on apoptosis but not on necrosis. These data show that the growth inhibitory and apoptosis-inducing effect of the saturated fatty acid palmitate on RINm5F cells is prevented by co-incubation with the polyunsaturated fatty acid linoleate but not inhibitors of ceramide or nitric oxide generation. A key role for phosphatidylinositol 3-kinase in mediating the linoleic-acid reduction in apoptosis is suggested.

Insulin‐like growth factor I reverses interleukin‐1β inhibition of insulin secretion, induction of nitric oxide synthase and cytokine‐mediated apoptosis in rat islets of Langerhans

We have previously observed that treatment of rat islets of Langerhans with interleukin‐1β for 12 h results in nitric oxide‐dependent inhibition of insulin secretion, while 48 h treatment increased rates of islet cell death by apoptosis. Here, we demonstrate that interleukin‐1β‐mediated nitric oxide formation and inhibition of insulin secretion are significantly reduced by 24 h pretreatment of rat islets of Langerhans with insulin‐like growth factor I (IGF‐I). IGF‐I decreased cytokine induction of nitric oxide synthase in islets. Use of an arginine analogue in culture or IGF‐I pretreatment of islets were also effective in protecting islets against cytokine‐mediated apoptotic cell death. We conclude that IGF‐I antagonises inhibitory and cytotoxic effects of cytokines in rat islets.

COMET ASSAY TO DETECT NITRIC OXIDE-DEPENDENT DNA DAMAGE IN MAMMALIAN CELLS

Publisher Summary This chapter discusses the use of comet assay to detect nitric oxide-dependent DNA damage in mammalian cells. After hydrogen peroxide, nitric oxide (NO) is the most prevalent mutagen to which human DNA is exposed. NO may interact with cellular amines to form N-nitroso compounds. Its oxidation products nitrite and the higher nitrogen oxides also have biological activity. NO produce DNA strand breakage in mammalian cells by the DNA precipitation assay, in situ nick translation, and the comet assay. The comet assay is a sensitive method for the detection of DNA strand breaks in mammalian cell. The comet assay detects release of DNA from a highly supercoiled DNA-protein complex. In comparison with other sensitive methods, the comet assay is relatively robust and economical in its use of material. It has the specific advantage that as a single-cell assay, it can detect nonuniform response within a cell population, and characterize the behavior of different cell types within a mixed population. Only a small proportion of DNA-damaging agents, including ionizing radiation, bleomycin, and hydrogen peroxide, induces direct breakage of the DNA phosphodiester backbone.