Bryndon J. Oleson

β-Cell Responses to Nitric Oxide

Autoimmune diabetes is characterized by the selective destruction of insulin-secreting β-cells that occurs during an inflammatory reaction in and around pancreatic islets of Langerhans. Cytokines such as interleukin-1, released by activated immune cells, have been shown to inhibit insulin secretion from pancreatic β-cells and cause islet destruction. In response to cytokines, β-cells express inducible nitric oxide synthase and produce micromolar levels of the free radical nitric oxide. Nitric oxide inhibits the mitochondrial oxidation of glucose resulting in an impairment of insulin secretion. Nitric oxide is also responsible for cytokine-mediated DNA damage in β-cells. While nitric oxide mediates the inhibitory and toxic effects of cytokines, it also activates protective pathways that allow β-cells to recover from this damage. This review will focus on the dual role of nitric oxide as a mediator of cytokine-induced damage and the activator of repair mechanisms that protect β-cells from cytokine-mediated injury.

Nitric Oxide Induces Ataxia Telangiectasia Mutated (ATM) Protein-dependent γH2AX Protein Formation in Pancreatic β Cells

Background: The mechanisms that control β cell fate following cytokine- and nitric oxide-induced damage remain unknown. Results: Cytokine-induced nitric oxide activates ATM and ATM-dependent caspase activation in β cells. Conclusion: ATM regulates the induction of apoptosis in cytokine-treated β cells. Significance: These studies define a role for DNA damage and ATM activation in nitric oxide-induced β cell apoptosis. In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic β cells. We show that cytokines stimulate H2AX phosphorylation (γH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated β cells.

Distinct differences in the responses of the human pancreatic β-cell line EndoC-βH1 and human islets to proinflammatory cytokines

While insulinoma cells have been developed and proven to be extremely useful in studies focused on mechanisms controlling β-cell function and viability, translating findings to human β-cells has proven difficult because of the limited access to human islets and the absence of suitable insulinoma cell lines of human origin. Recently, a human β-cell line, EndoC-βH1, has been derived from human fetal pancreatic buds. The purpose of this study was to determine whether human EndoC-βH1 cells respond to cytokines in a fashion comparable to human islets. Unlike most rodent-derived insulinoma cell lines that respond to cytokines in a manner consistent with rodent islets, EndoC-βH1 cells fail to respond to a combination of cytokines (IL-1, IFN-γ, and TNF) in a manner consistent with human islets. Nitric oxide, produced following inducible nitric oxide synthase (iNOS) expression, is a major mediator of cytokine-induced human islet cell damage. We show that EndoC-βH1 cells fail to express iNOS or produce nitric oxide in response to this combination of cytokines. Inhibitors of iNOS prevent cytokine-induced loss of human islet cell viability; however, they do not prevent cytokine-induced EndoC-βH1 cell death. Stressed human islets or human islets expressing heat shock protein 70 (HSP70) are resistant to cytokines, and, much like stressed human islets, EndoC-βH1 cells express HSP70 under basal conditions. Elevated basal expression of HSP70 in EndoC-βH1 cells is consistent with the lack of iNOS expression in response to cytokine treatment. While expressing HSP70, EndoC-βH1 cells fail to respond to endoplasmic reticulum stress activators, such as thapsigargin. These findings indicate that EndoC-βH1 cells do not faithfully recapitulate the response of human islets to cytokines. Therefore, caution should be exercised when making conclusions regarding the actions of cytokines on human islets when using this human-derived insulinoma cell line.

Inhibition of an NAD+ Salvage Pathway Provides Efficient and Selective Toxicity to Human Pluripotent Stem Cells

The tumorigenic potential of human pluripotent stem cells (hPSCs) is a major limitation to the widespread use of hPSC derivatives in the clinic. Here, we demonstrate that the small molecule STF‐31 is effective at eliminating undifferentiated hPSCs across a broad range of cell culture conditions with important advantages over previously described methods that target metabolic processes. Although STF‐31 was originally described as an inhibitor of glucose transporter 1, these data support the reclassification of STF‐31 as a specific NAD+ salvage pathway inhibitor through the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). These findings demonstrate the importance of an NAD+ salvage pathway in hPSC biology and describe how inhibition of NAMPT can effectively eliminate hPSCs from culture. These results will advance and accelerate the development of safe, clinically relevant hPSC‐derived cell‐based therapies.

How the location of superoxide generation influences the β-cell response to nitric oxide

Background: Although nitric oxide inhibits β-cell function, the impact of superoxide on nitric oxide signaling remains unknown. Results: Superoxide produced within β-cells inhibits nitric oxide-dependent responses, whereas extracellular generation does not. Conclusion: The reaction of nitric oxide with superoxide regulates the β-cell response to nitric oxide. Significance: The location of radical generation dictates the functional response of β-cells to reactive species. Cytokines impair the function and decrease the viability of insulin-producing β-cells by a pathway that requires the expression of inducible nitric oxide synthase (iNOS) and generation of high levels of nitric oxide. In addition to nitric oxide, excessive formation of reactive oxygen species, such as superoxide and hydrogen peroxide, has been shown to cause β-cell damage. Although the reaction of nitric oxide with superoxide results in the formation of peroxynitrite, we have shown that β-cells do not have the capacity to produce this powerful oxidant in response to cytokines. When β-cells are forced to generate peroxynitrite using nitric oxide donors and superoxide-generating redox cycling agents, superoxide scavenges nitric oxide and prevents the inhibitory and destructive actions of nitric oxide on mitochondrial oxidative metabolism and β-cell viability. In this study, we show that the β-cell response to nitric oxide is regulated by the location of superoxide generation. Nitric oxide freely diffuses through cell membranes, and it reacts with superoxide produced within cells and in the extracellular space, generating peroxynitrite. However, only when it is produced within cells does superoxide attenuate nitric oxide-induced mitochondrial dysfunction, gene expression, and toxicity. These findings suggest that the location of radical generation and the site of radical reactions are key determinants in the functional response of β-cells to reactive oxygen species and reactive nitrogen species. Although nitric oxide is freely diffusible, its biological function can be controlled by the local generation of superoxide, such that when this reaction occurs within β-cells, superoxide protects β-cells by scavenging nitric oxide.

Nitric Oxide Suppresses β-Cell Apoptosis by Inhibiting the DNA Damage Response.

ABSTRACT Nitric oxide, produced in pancreatic β cells in response to proinflammatory cytokines, plays a dual role in the regulation of β-cell fate. While nitric oxide induces cellular damage and impairs β-cell function, it also promotes β-cell survival through activation of protective pathways that promote β-cell recovery. In this study, we identify a novel mechanism in which nitric oxide prevents β-cell apoptosis by attenuating the DNA damage response (DDR). Nitric oxide suppresses activation of the DDR (as measured by γH2AX formation and the phosphorylation of KAP1 and p53) in response to multiple genotoxic agents, including camptothecin, H2O2, and nitric oxide itself, despite the presence of DNA damage. While camptothecin and H2O2 both induce DDR activation, nitric oxide suppresses only camptothecin-induced apoptosis and not H2O2-induced necrosis. The ability of nitric oxide to suppress the DDR appears to be selective for pancreatic β cells, as nitric oxide fails to inhibit DDR signaling in macrophages, hepatocytes, and fibroblasts, three additional cell types examined. While originally described as the damaging agent responsible for cytokine-induced β-cell death, these studies identify a novel role for nitric oxide as a protective molecule that promotes β-cell survival by suppressing DDR signaling and attenuating DNA damage-induced apoptosis.

Role of Protein Phosphatase 1 and Inhibitor of Protein Phosphatase 1 in Nitric Oxide–Dependent Inhibition of the DNA Damage Response in Pancreatic β-Cells

Nitric oxide is produced at micromolar levels by pancreatic β-cells during exposure to proinflammatory cytokines. While classically viewed as damaging, nitric oxide also activates pathways that promote β-cell survival. We have shown that nitric oxide, in a cell type–selective manner, inhibits the DNA damage response (DDR) and, in doing so, protects β-cells from DNA damage–induced apoptosis. This study explores potential mechanisms by which nitric oxide inhibits DDR signaling. We show that inhibition of DDR signaling (measured by γH2AX formation and the phosphorylation of KAP1) is selective for nitric oxide, as other forms of reactive oxygen/nitrogen species do not impair DDR signaling. The kinetics and broad range of DDR substrates that are inhibited suggest that protein phosphatase activation may be one mechanism by which nitric oxide attenuates DDR signaling in β-cells. While protein phosphatase 1 (PP1) is a primary regulator of DDR signaling and an inhibitor of PP1 (IPP1) is selectively expressed only in β-cells, disruption of either IPP1 or PP1 does not modify the inhibitory actions of nitric oxide on DDR signaling in β-cells. These findings support a PP1-independent mechanism by which nitric oxide selectively impairs DDR signaling and protects β-cells from DNA damage–induced apoptosis.