Hematologic presentation and the role of untargeted metabolomics analysis in monitoring treatment for riboflavin transporter deficiency.

Riboflavin transporter deficiency (RTD) (MIM #614707) is a neurogenetic disorder with its most common manifestations including sensorineural hearing loss, peripheral neuropathy, respiratory insufficiency, and bulbar palsy. Here, we present a 2-year-old boy whose initial presentation was severe macrocytic anemia necessitating multiple blood transfusions and intermittent neutropenia; he subsequently developed ataxia and dysarthria. Trio-exome sequencing detected compound heterozygous variants in SLC52A2 that were classified as pathogenic and a variant of uncertain significance. Bone marrow evaluation demonstrated megaloblastic changes. Notably, his anemia and neutropenia resolved after treatment with oral riboflavin, thus expanding the clinical phenotype of this disorder. We reiterate the importance of starting riboflavin supplementation in a young child who presents with macrocytic anemia and neurological features while awaiting biochemical and genetic work up. We detected multiple biochemical abnormalities with the help of untargeted metabolomics analysis associated with abnormal flavin adenine nucleotide function which normalized after treatment, emphasizing the reversible pathomechanisms involved in this disorder. The utility of untargeted metabolomics analysis to monitor the effects of riboflavin supplementation in RTD has not been previously reported.

Peroxiredoxin 1 plays a primary role in protecting pancreatic ß-cells from hydrogen peroxide and peroxynitrite.

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic ß-cell damage during the pathogenesis of autoimmune diabetes. While ß-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in ß-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of ß-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that ß-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 (Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 (Prdx2) had no effect. Together, these results suggest that ß-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.

The Role of Metabolic Flexibility in the Regulation of the DNA Damage Response by Nitric Oxide.

In this report, we show that nitric oxide suppresses DNA damage response (DDR) signaling in the pancreatic ß-cell line INS 832/13 and rat islets by inhibiting intermediary metabolism. Nitric oxide is known to inhibit complex IV of the electron transport chain and aconitase of the Krebs cycle. Non-ß cells compensate by increasing glycolytic metabolism to maintain ATP levels; however, ß cells lack this metabolic flexibility, resulting in a nitric oxide-dependent decrease in ATP and NAD+ Like nitric oxide, mitochondrial toxins inhibit DDR signaling in ß cells by a mechanism that is associated with a decrease in ATP. Non-ß cells compensate for the effects of mitochondrial toxins with an adaptive shift to glycolytic ATP generation that allows for DDR signaling. Forcing non-ß cells to derive ATP via mitochondrial respiration (replacing glucose with galactose in the medium) and glucose deprivation sensitizes these cells to nitric oxide-mediated inhibition of DDR signaling. These findings indicate that metabolic flexibility is necessary to maintain DDR signaling under conditions in which mitochondrial oxidative metabolism is inhibited and support the inhibition of oxidative metabolism (decreased ATP) as one protective mechanism by which nitric oxide attenuates DDR-dependent ß-cell apoptosis.

Pancreatic ß-cells detoxify H2O2 through the peroxiredoxin/thioredoxin antioxidant system.

Oxidative stress is thought to promote pancreatic ß-cell dysfunction and contribute to both type 1 and type 2 diabetes. Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are mediators of oxidative stress that arise largely from electron leakage during oxidative phosphorylation. Reports that ß-cells express low levels of antioxidant enzymes, including catalase and GSH peroxidases, have supported a model in which ß-cells are ill-equipped to detoxify ROS. This hypothesis seems at odds with the essential role of ß-cells in the control of metabolic homeostasis and organismal survival through exquisite coupling of oxidative phosphorylation, a prominent ROS-producing pathway, to insulin secretion. Using glucose oxidase to deliver H2O2 continuously over time and Amplex Red to measure extracellular H2O2 concentration, we found here that ß-cells can remove micromolar levels of this oxidant. This detoxification pathway utilizes the peroxiredoxin/thioredoxin antioxidant system, as selective chemical inhibition or siRNA-mediated depletion of thioredoxin reductase sensitized ß-cells to continuously generated H2O2 In contrast, when delivered as a bolus, H2O2 induced the DNA damage response, depleted cellular energy stores, and decreased ß-cell viability independently of thioredoxin reductase inhibition. These findings show that ß-cells have the capacity to detoxify micromolar levels of H2O2 through a thioredoxin reductase-dependent mechanism and are not as sensitive to oxidative damage as previously thought.

Decreased Sirtuin Deacetylase Activity in LRRK2 G2019S iPSC-Derived Dopaminergic Neurons.

Mitochondrial changes have long been implicated in the pathogenesis of Parkinson's disease (PD). The glycine to serine mutation (G2019S) in leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause for PD and has been shown to impair mitochondrial function and morphology in multiple model systems. We analyzed mitochondrial function in LRRK2 G2019S induced pluripotent stem cell (iPSC)-derived neurons to determine whether the G2019S mutation elicits similar mitochondrial deficits among central and peripheral nervous system neuron subtypes. LRRK2 G2019S iPSC-derived dopaminergic neuron cultures displayed unique abnormalities in mitochondrial distribution and trafficking, which corresponded to reduced sirtuin deacetylase activity and nicotinamide adenine dinucleotide levels despite increased sirtuin levels. These data indicate that mitochondrial deficits in the context of LRRK2 G2019S are not a global phenomenon and point to distinct sirtuin and bioenergetic deficiencies intrinsic to dopaminergic neurons, which may underlie dopaminergic neuron loss in PD.

Cardiomyocyte Differentiation Promotes Cell Survival During Nicotinamide Phosphoribosyltransferase Inhibition Through Increased Maintenance of Cellular Energy Stores.

To address concerns regarding the tumorigenic potential of undifferentiated human pluripotent stem cells (hPSC) that may remain after in vitro differentiation and ultimately limit the broad use of hPSC-derivatives for therapeutics, we recently described a method to selectively eliminate tumorigenic hPSC from their progeny by inhibiting nicotinamide phosphoribosyltransferase (NAMPT). Limited exposure to NAMPT inhibitors selectively removes hPSC from hPSC-derived cardiomyocytes (hPSC-CM) and spares a wide range of differentiated cell types; yet, it remains unclear when and how cells acquire resistance to NAMPT inhibition during differentiation. In this study, we examined the effects of NAMPT inhibition among multiple time points of cardiomyocyte differentiation. Overall, these studies show that in vitro cardiomyogenic commitment and continued culturing provides resistance to NAMPT inhibition and cell survival is associated with the ability to maintain cellular ATP pools despite depletion of NAD levels. Unlike cells at earlier stages of differentiation, day 28 hPSC-CM can survive longer periods of NAMPT inhibition and maintain ATP generation by glycolysis and/or mitochondrial respiration. This is distinct from terminally differentiated fibroblasts, which maintain mitochondrial respiration during NAMPT inhibition. Overall, these results provide new mechanistic insight into how regulation of cellular NAD and energy pools change with hPSC-CM differentiation and further inform how NAMPT inhibition strategies could be implemented within the context of cardiomyocyte differentiation. Stem Cells Translational Medicine 2017;6:1191-1201.

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

Nitric oxide, produced in pancreatic ß cells in response to proinflammatory cytokines, plays a dual role in the regulation of ß-cell fate. While nitric oxide induces cellular damage and impairs ß-cell function, it also promotes ß-cell survival through activation of protective pathways that promote ß-cell recovery. In this study, we identify a novel mechanism in which nitric oxide prevents ß-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 ß 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 ß-cell death, these studies identify a novel role for nitric oxide as a protective molecule that promotes ß-cell survival by suppressing DDR signaling and attenuating DNA damage-induced apoptosis.

Distinct differences in the responses of the human pancreatic ß-cell line EndoC-ßH1 and human islets to proinflammatory cytokines.

While insulinoma cells have been developed and proven to be extremely useful in studies focused on mechanisms controlling ß-cell function and viability, translating findings to human ß-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 ß-cell line, EndoC-ßH1, has been derived from human fetal pancreatic buds. The purpose of this study was to determine whether human EndoC-ßH1 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-ßH1 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-ßH1 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-ßH1 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-ßH1 cells express HSP70 under basal conditions. Elevated basal expression of HSP70 in EndoC-ßH1 cells is consistent with the lack of iNOS expression in response to cytokine treatment. While expressing HSP70, EndoC-ßH1 cells fail to respond to endoplasmic reticulum stress activators, such as thapsigargin. These findings indicate that EndoC-ßH1 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.

ATM facilitates mouse gammaherpesvirus reactivation from myeloid cells during chronic infection.

Gammaherpesviruses are cancer-associated pathogens that establish life-long infection in most adults. Insufficiency of Ataxia-Telangiectasia mutated (ATM) kinase leads to a poor control of chronic gammaherpesvirus infection via an unknown mechanism that likely involves a suboptimal antiviral response. In contrast to the phenotype in the intact host, ATM facilitates gammaherpesvirus reactivation and replication in vitro. We hypothesized that ATM mediates both pro- and antiviral activities to regulate chronic gammaherpesvirus infection in an immunocompetent host. To test the proposed proviral activity of ATM in vivo, we generated mice with ATM deficiency limited to myeloid cells. Myeloid-specific ATM deficiency attenuated gammaherpesvirus infection during the establishment of viral latency. The results of our study uncover a proviral role of ATM in the context of gammaherpesvirus infection in vivo and support a model where ATM combines pro- and antiviral functions to facilitate both gammaherpesvirus-specific T cell immune response and viral reactivation in vivo.

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 ß-cell response to nitric oxide.

Cytokines impair the function and decrease the viability of insulin-producing ß-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 ß-cell damage. Although the reaction of nitric oxide with superoxide results in the formation of peroxynitrite, we have shown that ß-cells do not have the capacity to produce this powerful oxidant in response to cytokines. When ß-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 ß-cell viability. In this study, we show that the ß-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 ß-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 ß-cells, superoxide protects ß-cells by scavenging nitric oxide.

Nitric oxide induces ataxia telangiectasia mutated (ATM) protein-dependent γH2AX protein formation in pancreatic ß cells.

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 ß 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 ß cells.

ß-Cell responses to nitric oxide.

Autoimmune diabetes is characterized by the selective destruction of insulin-secreting ß-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 ß-cells and cause islet destruction. In response to cytokines, ß-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 ß-cells. While nitric oxide mediates the inhibitory and toxic effects of cytokines, it also activates protective pathways that allow ß-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 ß-cells from cytokine-mediated injury.

S-Nitrosation of monocarboxylate transporter 1: inhibition of pyruvate-fueled respiration and proliferation of breast cancer cells.

Energy substrates metabolized through mitochondria (e.g., pyruvate, glutamine) are required for biosynthesis of macromolecules in proliferating cells. Because several mitochondrial proteins are known to be targets of S-nitrosation, we determined whether bioenergetics are modulated by S-nitrosation and defined the subsequent effects on proliferation. The nitrosating agent S-nitroso-L-cysteine (L-CysNO) was used to initiate intracellular S-nitrosation, and treatment decreased mitochondrial function and inhibited proliferation of MCF7 mammary adenocarcinoma cells. Surprisingly, the d-isomer of CysNO (D-CysNO), which is not transported into cells, also caused mitochondrial dysfunction and limited proliferation. Both L- and D-CysNO also inhibited cellular pyruvate uptake and caused S-nitrosation of thiol groups on monocarboxylate transporter 1, a proton-linked pyruvate transporter. These data demonstrate the importance of mitochondrial metabolism in proliferative responses in breast cancer and highlight a novel role for inhibition of metabolic substrate uptake through S-nitrosation of exofacial protein thiols in cellular responses to nitrosative stress.

Inducible HSP70 regulates superoxide dismutase-2 and mitochondrial oxidative stress in the endothelial cells from developing lungs.

Superoxide dismutase 2 (SOD-2) is synthesized in the cytosol and imported into the mitochondrial matrix, where it is activated and functions as the primary antioxidant for cellular respiration. The specific mechanisms that target SOD-2 to the mitochondria remain unclear. We hypothesize that inducible heat shock protein 70 (iHSP70) targets SOD-2 to the mitochondria via a mechanism facilitated by ATP, and this process is impaired in persistent pulmonary hypertension of the newborn (PPHN). We observed that iHSP70 interacts with SOD-2 and targets SOD-2 to the mitochondria. Interruption of iHSP70-SOD-2 interaction with 2-phenylethylenesulfonamide-µ (PFT-µ, a specific inhibitor of substrate binding to iHSP70 COOH terminus) and siRNA-mediated knockdown of iHSP70 expression disrupted SOD-2 transport to mitochondria. Increasing intracellular ATP levels by stimulation of respiration with CaCl2 facilitated the mitochondrial import of SOD-2, increased SOD-2 activity, and decreased the mitochondrial superoxide (O2(·-)) levels in PPHN pulmonary artery endothelial cells (PAEC) by promoting iHSP70-SOD-2 dissociation at the outer mitochondrial membrane. In contrast, oligomycin, an inhibitor of mitochondrial ATPase, decreased SOD-2 expression and activity and increased O2(·-) levels in the mitochondria of control PAEC. The basal ATP levels and degree of iHSP70-SOD-2 dissociation were lower in PPHN PAEC and lead to increased SOD-2 degradation in cytosol. In normal pulmonary arteries (PA), PFT-µ impaired the relaxation response of PA rings in response to nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine. Pretreatment with Mito-Q, a mitochondrial targeted O2(·-) scavenger, restored the relaxation response in PA rings pretreated with PFT-µ. Our observations suggest that iHSP70 chaperones SOD-2 to the mitochondria. Impaired SOD-2-iHSP70 dissociation decreases SOD-2 import and contributes to mitochondrial oxidative stress in PPHN.

Do ß-cells generate peroxynitrite in response to cytokine treatment?

The purpose of this study was to determine the reactive species that is responsible for cytokine-mediated ß-cell death. Inhibitors of inducible nitric oxide synthase prevent this death, and addition of exogenous nitric oxide using donors induces ß-cell death. The reaction of nitric oxide with superoxide results in the generation of peroxynitrite, and this powerful oxidant has been suggested to be the mediator of ß-cell death in response to cytokine treatment. Recently, coumarin-7-boronate has been developed as a probe for the selective detection of peroxynitrite. Using this reagent, we show that addition of the NADPH oxidase activator phorbol 12-myristate 13-acetate to nitric oxide-producing macrophages results in peroxynitrite generation. Using a similar approach, we demonstrate that cytokines fail to stimulate peroxynitrite generation by rat islets and insulinoma cells, either with or without phorbol 12-myristate 13-acetate treatment. When forced to produce superoxide using redox cyclers, this generation is associated with protection from nitric oxide toxicity. These findings indicate that: (i) nitric oxide is the likely mediator of the toxic effects of cytokines, (ii) ß-cells do not produce peroxynitrite in response to cytokines, and (iii) when forced to produce superoxide, the scavenging of nitric oxide by superoxide is associated with protection of ß-cells from nitric oxide-mediated toxicity.

Nitrosative stress and redox-cycling agents synergize to cause mitochondrial dysfunction and cell death in endothelial cells.

Nitric oxide production by the endothelium is required for normal vascular homeostasis; however, in conditions of oxidative stress, interactions of nitric oxide with reactive oxygen species (ROS) are thought to underlie endothelial dysfunction. Beyond canonical nitric oxide signaling pathways, nitric oxide production results in the post-translational modification of protein thiols, termed S-nitrosation. The potential interplay between S-nitrosation and ROS remains poorly understood and is the focus of the current study. The effects of the S-nitrosating agent S-nitrosocysteine (CysNO) in combination with redox-cycling agents was examined in bovine aortic endothelial cells (BAEC). CysNO significantly impairs mitochondrial function and depletes the NADH/NAD(+) pool; however, these changes do not result in cell death. When faced with the additional stressor of a redox-cycling agent used to generate ROS, further loss of NAD(+) occurs, and cellular ATP pools are depleted. Cellular S-nitrosothiols also accumulate, and cell death is triggered. These data demonstrate that CysNO sensitizes endothelial cells to redox-cycling agent-dependent mitochondrial dysfunction and cell death and identify attenuated degradation of S-nitrosothiols as one potential mechanism for the enhanced cytotoxicity.

Effect of nitric oxide on naphthoquinone toxicity in endothelial cells: role of bioenergetic dysfunction and poly (ADP-ribose) polymerase activation.

When produced at physiological levels, reactive oxygen species (ROS) can act as signaling molecules to regulate normal vascular function. Produced under pathological conditions, ROS can contribute to the oxidative damage of cellular components (e.g., DNA and proteins) and trigger cell death. Moreover, the reaction of superoxide with nitric oxide (NO) produces the strong oxidant peroxynitrite and decreases NO bioavailability, both of which may contribute to activation of cell death pathways. The effects of ROS generated from the 1,4-naphthoquinones alone and in combination with NO on the activation status of poly(ADP-ribose) polymerase (PARP) and cell viability were examined. Treatment with redox cycling quinones activates PARP, and this stimulatory effect is attenuated in the presence of NO. Mitochondria play a central role in cell death signaling pathways and are a target of oxidants. We show that simultaneous exposure of endothelial cells to NO and ROS results in mitochondrial dysfunction, ATP and NAD(+) depletion, and cell death. Alone, NO and ROS have only minor effects on cellular bioenergetics. Further, PARP inhibition does not attenuate reduced cell viability or mitochondrial dysfunction. These results show that concomitant exposure to NO and ROS impairs energy metabolism and triggers PARP-independent cell death. While superoxide-mediated PARP activation is attenuated in the presence of NO, PARP inhibition does not modify the loss of mitochondrial function or adenine and pyridine nucleotide pools and subsequent bioenergetic dysfunction. These findings suggest that the mechanisms by which ROS and NO induce endothelial cell death are closely linked to the maintenance of mitochondrial function and not overactivation of PARP.