Hughes KJ, Chambers KT, Meares GP, Corbett JA. Nitric oxides mediates a shift from early necrosis to late apoptosis in cytokine-treated ␤-cells that is associated with irreversible DNA damage. Am J Physiol Endocrinol Metab 297: E1187-E1196, 2009. First published September 8, 2009 doi:10.1152/ajpendo.00214.2009.-For many cell types, including pancreatic ␤-cells, nitric oxide is a mediator of cell death; however, it is paradoxical that for a given cell type nitric oxide can induce both necrosis and apoptosis. This report tests the hypothesis that cell death mediated by nitric oxide shifts from an early necrotic to a late apoptotic event. Central to this transition is the ability of ␤-cells to respond and repair nitric oxide-mediated damage. ␤-Cells have the ability to repair DNA that is damaged following 24-h incubation with IL-1; however, cytokine-induced DNA damage becomes irreversible following 36-h incubation. This irreversible DNA damage following 36-h incubation with IL-1 correlates with the activation of caspase-3 (cleavage and activity). The increase in caspase activity correlates with reductions in endogenous nitric oxide production, as nitric oxide is an inhibitor of caspase activity. In contrast, caspase cleavage or activation is not observed under conditions in which ␤-cells are capable of repairing damaged DNA (24-h incubation with cytokines). These findings provide evidence that ␤-cell death in response to cytokines shifts from an early necrotic process to apoptosis and that this shift is associated with irreversible DNA damage and caspase-3 activation.islet; insulin; death INSULIN-DEPENDENT DIABETES MELLITUS is an autoimmune disease characterized by the selective destruction of pancreatic ␤-cells found in islets of Langerhans (20). Cytokines, released by invading leukocytes during insulitis, are believed to play a role in ␤-cell destruction (32, 41). In rodent islets, the macrophagederived cytokine interleukin (IL)-1 is sufficient to impair insulin secretion and induce islet damage (10). The destructive actions of IL-1 are augmented by IFN␥ and TNF (50). In islets isolated from most mouse strains and humans, IL-1 and IFN␥ are the minimal combination of cytokines required to induce damage, and this damage is enhanced by TNF (2,13,23,33).Nitric oxide plays a central role in regulating the response(s) of ␤-cells to cytokines. Cytokines stimulate the expression of the inducible isoform of nitric oxide synthase (iNOS) and the production of micromolar levels of nitric oxide by ␤-cells (9,11,15,17,48). Nitric oxide reduces ␤-cell viability, as determined by the neutral red assay as well as 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (49). In addition, nitric oxide inhibits insulin secretion by attenuating the oxidation of glucose to CO 2 by inhibiting the activity of mitochondrial iron-sulfur center-containing enzymes such as aconitase and complexes of the electron transport system (15). This results in a fourfold decrease in cellular ATP levels (18, 30). Since glucose-sti...

For many cell types, including pancreatic ␤-cells, nitric oxide is a mediator of cell death; paradoxically, nitric oxide can also activate pathways that promote the repair of cellular damage. In this report, a role for FoxO1-dependent transcriptional activation and its regulation by SIRT1 in determining the cellular response to nitric oxide is provided. In response to nitric oxide, FoxO1 translocates from the cytoplasm to the nucleus and stimulates the expression of the DNA repair gene GADD45␣, resulting in FoxO1-dependent DNA repair. FoxO1-dependent gene expression appears to be regulated by the NAD ؉ -dependent deacetylase SIRT1. In response to SIRT1 inhibitors, the FoxO1-dependent protective actions of nitric oxide (GADD45␣ expression and DNA repair) are attenuated, and FoxO1 activates a proapoptotic program that includes PUMA (p53-up-regulated mediator of apoptosis) mRNA accumulation and caspase-3 cleavage. These findings support primary roles for FoxO1 and SIRT1 in regulating the cellular responses of ␤-cells to nitric oxide.Nitric oxide plays a central role in regulating the response(s) of pancreatic ␤-cells to cytokine treatment. Cytokines such as IL-1 (rat) and a combination of IL-1 ϩ IFN-␥ (mouse and human) stimulate the expression of the inducible isoform of nitric-oxide synthase (NOS) and the production of micromolar levels of nitric oxide by ␤-cells (1-4). Nitric oxide attenuates insulin secretion by inhibiting the oxidation of glucose to CO 2 and the activity of mitochondrial iron-sulfur center containing enzymes such as aconitase and complexes of the electron transport system (5, 6). The result is a 4-fold reduction in cellular ATP concentration (2, 7) that leads to the inhibition of glucose-induced insulin secretion due to the inability to generate sufficient levels of ATP to close the ATPsensitive K ϩ channels, an event required for ␤-cell depolarization and Ca 2ϩ -dependent exocytosis (8, 9). In addition to the inhibition of ␤-cell function, nitric oxide induces DNA strand breaks and oxidative DNA damage (10, 11).The inhibitory actions of IL-1 on ␤-cell function and DNA damage are reversible (12, 13). The addition of a NOS inhibitor to islets pretreated for 24 h with IL-1 (without removing IL-1) results in a time-dependent recovery of insulin secretion and mitochondrial function (3) and the repair of damaged DNA (14,15). This recovery response requires new gene expression, the activation of JNK, and can be stimulated by nitric oxide (16,17). The ability of ␤-cells to recover from cytokine-and nitric oxide-induced damage is temporally limited. Following a 36-h exposure to IL-1, ␤-cells are no longer capable of recovering metabolic and secretory function, and the islets are committed to death (14, 18). Studies have shown that cytokines can kill ␤-cells by nitric oxide-dependent and -independent necrotic and apoptotic mechanisms (19 -26). This dichotomy in the type of cell death that has been observed appears to reflect the temporal changes in the metabolic responses and the extent of DNA damage ca...

Proinflammatory cytokines induce nitric oxide-dependent DNA damage and ultimately ␤-cell death. Not only does nitric oxide cause ␤-cell damage, it also activates a functional repair process. In this study, the mechanisms activated by nitric oxide that facilitate the repair of damaged ␤-cell DNA are examined. JNK plays a central regulatory role because inhibition of this kinase attenuates the repair of nitric oxide-induced DNA damage. p53 is a logical target of JNK-dependent DNA repair; however, nitric oxide does not stimulate p53 activation or accumulation in ␤-cells. Further, knockdown of basal p53 levels does not affect DNA repair. In contrast, expression of growth arrest and DNA damage (GADD) 45␣, a DNA repair gene that can be regulated by p53-dependent and p53-independent pathways, is stimulated by nitric oxide in a JNK-dependent manner, and knockdown of GADD45␣ expression attenuates the repair of nitric oxide-induced ␤-cell DNA damage. These findings show that ␤-cells have the ability to repair nitric oxide-damaged DNA and that JNK and GADD45␣ mediate the p53-independent repair of this DNA damage.Insulin-dependent diabetes mellitus is an autoimmune disease characterized by the selective destruction of insulin-secreting pancreatic ␤-cells found in the islets of Langerhans (1). Cytokines, released from invading leukocytes during insulitis, are believed to participate in the initial destruction of ␤-cells, precipitating the autoimmune response (2, 3). Treatment of rat islets with the macrophage-derived cytokine interleukin-1 (IL -1) 2 results in the inhibition of glucose-stimulated insulin secretion and oxidative metabolism and in the induction of DNA damage that ultimately results in ␤-cell death (4 -6). Nitric oxide, produced in micromolar levels following enhanced expression of the inducible nitric-oxide synthase in ␤-cells, mediates the damaging actions of cytokines on ␤-cell function (7-9). Nitric oxide inhibits insulin secretion by attenuating the oxidation of glucose to CO 2 , reducing cellular levels of ATP and, thereby, attenuating ATP-inhibited K ϩ channel activity (10, 11). The net effect is the inhibition of ␤-cell depolarization, calcium entry, and calcium-dependent exocytosis. In addition to the inhibition of ␤-cell function, nitric oxide induces DNA damage in ␤-cells (4, 12, 13). Nitric oxide or the oxidation products N 2 O 3 and ONOO Ϫ induce DNA damage through direct strand breaks and base modification (14 -16) and by inhibition of DNA repair enzymes, thereby enhancing the damaging actions of nitric oxide (17,18).Recent studies have shown that ␤-cells maintain a limited ability to recover from cytokine-mediated damage (19,20). The addition of a nitric-oxide synthase inhibitor to islets treated for 24 h with cytokine and continued culture with the nitric-oxide synthase inhibitor and cytokine results in a time-dependent restoration of insulin secretion, mitochondrial aconitase activity, and the repair of nitric oxide-damaged DNA (20, 21). Nitric oxide plays a dual role in modifying ␤-cell respon...

While there can be detrimental consequences of nitric oxide production at pathological concentrations, eukaryotic cells have evolved protective mechanisms to defend themselves against this damage. The unfoldedprotein response (UPR), activated by misfolded proteins and oxidative stress, is one adaptive mechanism that is employed to protect cells from stress. Nitric oxide is a potent activator of AMP-activated protein kinase (AMPK), and AMPK participates in the cellular defense against nitric oxide-mediated damage in pancreatic ␤-cells. In this study, the mechanism of AMPK activation by nitric oxide was explored. The known AMPK kinases LKB1, CaMKK, and TAK1 are not required for the activation of AMPK by nitric oxide. Instead, this activation is dependent on the endoplasmic reticulum (ER) stress-activated protein IRE1. Nitric oxide-induced AMPK phosphorylation and subsequent signaling to AMPK substrates, including Raptor, acetyl coenzyme A carboxylase, and PGC-1␣, is attenuated in IRE1␣-deficient cells. The endoribonuclease activity of IRE1 appears to be required for AMPK activation in response to nitric oxide. In addition to nitric oxide, stimulation of IRE1 endoribonuclease activity with the flavonol quercetin leads to IRE1-dependent AMPK activation. These findings indicate that the RNase activity of IRE1 participates in AMPK activation and subsequent signaling through multiple AMPK-dependent pathways in response to nitrosative stress.Nitric oxide, an important mediator of both physiological and pathological processes, has been implicated in the development of a number of inflammatory diseases. When produced at low concentrations, nitric oxide can promote cell growth and survival. At high concentrations, such as those produced during inflammation by inducible nitric oxide synthase (iNOS), nitric oxide induces extensive cellular injury that includes DNA damage, inhibition of oxidative metabolism, and induction of endoplasmic reticulum (ER) stress (5, 12, 39). Pancreatic ␤-cells are exquisitely sensitive to oxidative damage, as glucose-stimulated insulin secretion requires the oxidation of glucose to CO 2 , resulting in the accumulation of ATP. Nitric oxide, produced in micromolar concentrations in response to interleukin 1 (IL-1) and gamma interferon (IFN-␥), mediates the damaging effects of these cytokines on ␤-cell function (3, 33). While nitric oxide stimulates cellular damage, it also activates a number of signaling pathways that limit additional cellular damage and repair existing damage. In pancreatic ␤-cells, the protective responses activated by nitric oxide include (i) JNK-dependent induction of GADD45␣ (growth arrest and DNA damage-inducible protein 45␣) and DNA repair, (ii) activation of AMP-activated protein kinase (AMPK), resulting in enhanced metabolic recovery, and (iii) activation of the unfolded-protein response (UPR) (25,34,38,54,57,61).AMPK is a conserved heterotrimeric (␣, ␤, and ␥ subunits) serine/threonine kinase involved in sensing and responding to the energetic demand within eukaryotic cel...