Repurposing FDA-approved drugs to treat chemical weapon toxicities: Interactive case studies for trainees.

The risk of a terrorist attack in the United States has created challenges on how to effectively treat toxicities that result from exposure to chemical weapons. To address this concern, the United States has organized a trans-agency initiative across academia, government, and industry to identify drugs to treat tissue injury resulting from exposure to chemical threat agents. We sought to develop and evaluate an interactive educational session that provides hands-on instruction on how to repurpose FDA-approved drugs as therapeutics to treat toxicity from exposure to chemical weapons. As part of the Rutgers Summer Undergraduate Research Fellowship program, 23 undergraduate students participated in a 2-h session that included: (1) an overview of chemical weapon toxicities, (2) a primer on pharmacology principles, and (3) an interactive session where groups of students were provided lists of FDA-approved drugs to evaluate potential mechanisms of action and suitability as countermeasures for four chemical weapon case scenarios. The interactive session culminated in a competition for the best grant "sales pitch." From this interactive training, students improved their understanding of (1) the ability of chemical weapons to cause long-term toxicities, (2) impact of route of administration and exposure scenario on drug efficacy, and (3) re-purposing FDA-approved drugs to treat disease from chemical weapon exposure. These findings demonstrated that an interactive training exercise can provide students with new insights into drug development for chemical threat agent toxicities.

Wastewater Surveillance of US Coast Guard Installations and Seagoing Military Vessels to Mitigate the Risk of COVID-19 Outbreaks, March 2021-August 2022.

OBJECTIVES: Military training centers and seagoing vessels are often environments at high risk for the spread of COVID-19 and other contagious diseases, because military trainees and personnel arrive after traveling from many parts of the country and live in congregate settings. We examined whether levels of SARS-CoV-2 genetic material in wastewater correlated with SARS-CoV-2 infections among military personnel living in communal barracks and vessels at US Coast Guard training centers in the United States. METHODS: The Coast Guard developed and established 3 laboratories with wastewater testing capability at Coast Guard training centers from March 2021 through August 2022. We analyzed wastewater from barracks housing trainees and from 4 Coast Guard vessels for the presence of SARS-CoV-2 genes N and E and quantified the results relative to levels of a fecal indicator virus, pepper mild mottle virus. We compared quantified data with the timing of medically diagnosed COVID-19 infection among (1) military personnel who had presented with symptoms or had been discovered through contact tracing and had medical tests and (2) military personnel who had been discovered through routine surveillance by positive SARS-CoV-2 antigen or polymerase chain reaction test results. RESULTS: Levels of viral genes in wastewater at Coast Guard locations were best correlated with diagnosed COVID-19 cases when wastewater testing was performed twice weekly with passive samplers deployed for the entire week; such testing detected ≥1 COVID-19 case 69.8% of the time and ≥3 cases 88.3% of the time. Wastewater assessment in vessels did not continue because of logistical constraints. CONCLUSION: Wastewater testing is an effective tool for measuring the presence and patterns of SARS-CoV-2 infections among military populations. Success with wastewater testing for SARS-CoV-2 infections suggests that other diseases may be assessed with similar approaches.

DIRAS3 induces autophagy and enhances sensitivity to anti-autophagic therapy in KRAS-driven pancreatic and ovarian carcinomas.

Pancreatic ductal adenocarcinoma (PDAC) and low-grade ovarian cancer (LGSOC) are characterized by the prevalence of KRAS oncogene mutations. DIRAS3 is the first endogenous non-RAS protein that heterodimerizes with RAS, disrupts RAS clustering, blocks RAS signaling, and inhibits cancer cell growth. Here, we found that DIRAS3-mediated KRAS inhibition induces ROS-mediated apoptosis in PDAC and LGSOC cells with KRAS mutations, but not in cells with wild-type KRAS, by downregulating NFE2L2/Nrf2 transcription, reducing antioxidants, and inducing oxidative stress. DIRAS3 also induces cytoprotective macroautophagy/autophagy that may protect mutant KRAS cancer cells from oxidative stress, by inhibiting mutant KRAS, activating the STK11/LKB1-PRKAA/AMPK pathway, increasing lysosomal CDKN1B/p27 localization, and inducing autophagic gene expression. Treatment with chloroquine or the novel dimeric chloroquine analog DC661 significantly enhances DIRAS3-mediated inhibition of mutant KRAS tumor cell growth in vitro and in vivo. Taken together, our study demonstrates that DIRAS3 plays a critical role in regulating mutant KRAS-driven oncogenesis in PDAC and LGSOC.Abbreviations: AFR: autophagic flux reporter; ATG: autophagy related; CQ: chloroquine; DCFDA: 2'-7'-dichlorodihydrofluorescein diacetate; DIRAS3: DIRAS family GTPase 3; DOX: doxycycline; KRAS: KRAS proto-oncogene, LGSOC: low-grade serous ovarian cancer; MiT/TFE: microphthalmia family of transcription factors; NAC: N-acetylcysteine; PDAC: pancreatic ductal adenocarcinoma; ROS: reactive oxygen species; TFEB: transcription factor EB.

Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function.

DIRAS3 is an imprinted tumor suppressor gene encoding a GTPase that has a distinctive N-terminal extension (NTE) not found in other RAS proteins. This NTE and the prenylated C-terminus are required for DIRAS3-mediated inhibition of RAS/MAP signaling and PI3K activity at the plasma membrane. In this study, we applied biochemical, biophysical, and computational methods to characterize the structure and function of the NTE. The NTE peptide recognizes phosphoinositides PI(3,4,5)P3 and PI(4,5)P2 with rapid kinetics and strong affinity. Lipid binding induces NTE structural change from disorder to amphipathic helix. Mass spectrometry identified N-myristoylation of DIRAS3. All-atom molecular dynamic simulations predict DIRAS3 could adhere to the membrane through both termini, suggesting the NTE is involved in targeting and stabilizing DIRAS3 on the membrane by double anchoring. Overall, our results are consistent with DIRAS3's function as a tumor suppressor, whereby the membrane-bound DIRAS3 can effectively target PI3K and KRAS at the membrane.

Directed evolution of cyclic peptides for inhibition of autophagy.

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B-two essential components of the autophagosome maturation machinery-with mid-nanomolar affinities and disrupted protein-protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

The Strategic National Stockpile: identification, support, and acquisition of medical countermeasures for CBRN incidents.

The Strategic National Stockpile (SNS) serves as a repository of materiel, including medical countermeasures (MCMs), that would be used to support the national health security response to a chemical, biological, radiological, or nuclear (CBRN) incident, either natural or terrorism-related. To support and advance the SNS, the National Institutes of Health (NIH) manages targeted investigatory research portfolios, such as Countermeasures Against Chemical Terrorism (CounterACT) for chemical agents, that coordinate projects covering basic research, drug discovery, and preclinical studies. Project BioShield, managed by the Biomedical Advanced Research and Development Agency (BARDA), guides and supports academia and industry with potential MCMs through the Food & Drug Administration's approval process and ultimately supports the acquisition of successful products into the SNS. Public health emergencies such as the COVID-19 pandemic and the ever-increasing number of MCMs in the SNS present logistical and financial challenges to its maintenance. While MCMs for biological agents have been readily adopted, those for chemical agents have required sustained investments. This paper reviews the methods by which MCMs are identified and supported for inclusion in the SNS, the current status of MCMs for CBRN threats, and challenges with SNS maintenance as well as identifies persistent obstacles for MCM development and acquisition, particularly for ones focused on chemical weapons.

Purification of poly-dA oligonucleotides and mRNA-protein fusions with dT25-OAS resin.

Solid-phase resins functionalized with poly-deoxythymidine (dT) oligos facilitate purification of poly-adenylated molecules from solution through high affinity, high selectivity base-pairing interactions. These resins are commonly used to purify messenger RNA (mRNA) from complex biological mixtures as well as mRNA-protein fusion molecules for mRNA Display selections. Historically, dT-conjugated cellulose was the primary resin for poly-dA purification, but its scarcity has prompted the development of alternative resins, most notably dT-functionalized magnetic beads. In order to develop a cost-effective alternative to commercially available poly-dT resins for large-scale purifications of mRNA-protein fusions, we investigated the purification properties of dT25-conjugated Oligo Affinity Support resin (dT25-OAS) alongside poly-dT14 magnetic beads and dT25-cellulose. dT25-OAS was found to have the highest dA21 oligo binding capacity at 4 pmol/µg, followed by dT14-magnetic beads (1.1 pmol/µg) and dT25-cellulose (0.7 pmol/µg). To determine the resin specificity in the context of a complex biological mixture, we translated mRNA-protein fusions consisting of a radiolabeled Her2 affibody fused to its encoding mRNA. Commercial dT25-cellulose showed the highest mRNA-affibody purification specificity, followed by dT25-OAS and dT14-magnetic beads. Overall, dT25-OAS showed exceptionally high binding capacity and low background binding, making it an attractive alternative for large-scale mRNA purification and mRNA Display library enrichment.

Society of Toxicology Develops Learning Framework for Undergraduate Toxicology Courses Following the Vision and Change Core Concepts Model.

The Society of Toxicology announces the development of a Learning Framework (https://www.toxicology.org/education/docs/SOT-Toxicology-Learning-Objectives.pdf) for undergraduate toxicology that will facilitate the development and sharing of evidence-based teaching materials for undergraduate toxicology educators throughout the world. This Learning Framework was modeled on the "Vision and Change Report" (www.visionandchange.org), an effort of the National Science Foundation and American Association for the Advancement of Science defining Core Concepts and Core Competencies to inform undergraduate biology course design. Vision and Change (V&C) has gained national acceptance, becoming a foundation for 14 upper-level courses designed by professional life science scientific societies. The undergraduate toxicology Learning Framework includes 5 Core Concepts aligned with V&C that encompass the discipline of toxicology: Evolution; Biological Information, Risk and Risk Management; Systems Toxicology; and Pathways and Transformations for Energy and Matter. Underpinning the Core Concepts are Level 2 Toxicology Concepts, which are broad disciplinary categories, Level 3 Learning Objectives, which address specific learning goals, and Level 4 Example Learning Objectives and Case Studies, which provide examples of how content might be taught. Syllabi from more than 20 undergraduate toxicology courses and several undergraduate toxicology textbooks were surveyed to determine toxicology-related Learning Objectives. From these, undergraduate educators can design courses tailored to their institutional needs by selecting a subset of Learning Objectives. Publication of a Learning Framework for toxicology will enable integration into other disciplines and facilitate the development and sharing of evidenced-based teaching materials for toxicology to educators in allied disciplines. Ultimately this will expand toxicology's impact to a broader audience.

DIRAS3-Derived Peptide Inhibits Autophagy in Ovarian Cancer Cells by Binding to Beclin1.

Autophagy can protect cancer cells from acute starvation and enhance resistance to chemotherapy. Previously, we reported that autophagy plays a critical role in the survival of dormant, drug resistant ovarian cancer cells using human xenograft models and correlated the up-regulation of autophagy and DIRAS3 expression in clinical samples obtained during "second look" operations. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26 kD GTPase with homology to RAS that inhibits cancer cell proliferation and motility. Re-expression of DIRAS3 in ovarian cancer xenografts also induces dormancy and autophagy. DIRAS3 can bind to Beclin1 forming the Autophagy Initiation Complex that triggers autophagosome formation. Both the N-terminus of DIRAS3 (residues 15-33) and the switch II region of DIRAS3 (residues 93-107) interact directly with BECN1. We have identified an autophagy-inhibiting peptide based on the switch II region of DIRAS3 linked to Tat peptide that is taken up by ovarian cancer cells, binds Beclin1 and inhibits starvation-induced DIRAS3-mediated autophagy.

Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers.

Radiolabeling of substrates with 2-[18F]fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide-alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the 18F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors >500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[18F]fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/µmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time.

NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) and cytochrome P450 oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic ß-cells.

NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic ß-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of ß-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H2O2. Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in ß-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in ß-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic ß-cells.

Mechanisms of Doxorubicin Toxicity in Pancreatic ß-Cells.

Exposure to chemotherapeutic agents has been linked to an increased risk of type 2 diabetes (T2D), a disease characterized by both the peripheral insulin resistance and impaired glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells. Using the rat ß-cell line INS-1 832/13 and isolated mouse pancreatic islets, we investigated the effect of the chemotherapeutic drug doxorubicin (Adriamycin) on pancreatic ß-cell survival and function. Exposure of INS-1 832/13 cells to doxorubicin caused impairment of GSIS, cellular viability, an increase in cellular toxicity, as soon as 6 h post-exposure. Doxorubicin impaired plasma membrane electron transport (PMET), a pathway dependent on reduced equivalents NADH and NADPH, but failed to redox cycle in INS-1 832/13 cells and with their lysates. Although NADPH/NADP(+ )content was unaffected, NADH/NAD(+ )content decreased at 4 h post-exposure to doxorubicin, and was followed by a reduction in ATP content. Previous studies have demonstrated that doxorubicin functions as a topoisomerase II inhibitor via induction of DNA cross-linking, resulting in apoptosis. Doxorubicin induced the expression of mRNA for mdm2, cyclin G1, and fas whereas downregulating p53, and increased the melting temperature of genomic DNA, consistent with DNA damage and induction of apoptosis. Doxorubicin also induced caspase-3 and -7 activity in INS-1 832/13 cells and mouse islets; co-treatment with the pan-caspase inhibitor Z-VAD-FMK temporarily attenuated the doxorubicin-mediated loss of viability in INS-1 832/13 cells. Together, these data suggest that DNA damage, not H2O2 produced via redox cycling, is a major mechanism of doxorubicin toxicity in pancreatic ß-cells.

Thymoquinone, a bioactive component of Nigella sativa, normalizes insulin secretion from pancreatic ß-cells under glucose overload via regulation of malonyl-CoA.

Thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone) is a major bioactive component of Nigella sativa, a plant used in traditional medicine to treat a variety of symptoms, including elevated blood glucose levels in type 2 diabetic patients. Normalization of elevated blood glucose depends on both glucose disposal by peripheral tissues and glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells. We employed clonal ß-cells and rodent islets to investigate the effects of thymoquinone (TQ) and Nigella sativa extracts (NSEs) on GSIS and cataplerotic metabolic pathways implicated in the regulation of GSIS. TQ and NSE regulated NAD(P)H/NAD(P)(+) ratios via a quinone-dependent redox cycling mechanism. TQ content was positively correlated with the degree of redox cycling activity of NSE extracts, suggesting that TQ is a major component engaged in mediating NSE-dependent redox cycling. Both acute and chronic exposure to TQ and NSE enhanced GSIS and were associated with the ability of TQ and NSE to increase the ATP/ADP ratio. Furthermore, TQ ameliorated the impairment of GSIS following chronic exposure of ß-cells to glucose overload. This protective action was associated with the TQ-dependent normalization of chronic accumulation of malonyl-CoA, elevation of acetyl-CoA carboxylase (ACC), fatty acid synthase, and fatty acid-binding proteins following chronic glucose overload. Together, these data suggest that TQ modulates the ß-cell redox circuitry and enhances the sensitivity of ß-cell metabolic pathways to glucose and GSIS under normal conditions as well as under hyperglycemia. This action is associated with the ability of TQ to regulate carbohydrate-to-lipid flux via downregulation of ACC and malonyl-CoA.

Curcumin, Hesperidin, and Rutin Selectively Interfere with Apoptosis Signaling and Attenuate Streptozotocin-Induced Oxidative Stress-Mediated Hyperglycemia.

Type I Diabetes is characterized by the presence of hyperglycemia due to insulin deficiency and consequent impaired hepatic glucose metabolism. During diabetes, the liver becomes the most important tissue for the regulation of serum glucose. However, elevated glucose causes continuous oxidative damage to the liver, reducing its capacity to ameliorate hyperglycemia, which contributes to macrovascular complications [1]. Numerous epidemiological studies have demonstrated that excess human consumption of diets rich in specific bioflavonoid phytochemicals attenuates the effects of diabetes. Thus, this study was designed to investigate whether a bioflavonoid mixture could : i) attenuate streptozotocin (STZ)-induced hyperglycemia, ii) potentiate antioxidant signaling in the liver, and iii) ameliorate the apoptotic signaling cascade in the liver of STZ-induced hyperglycemic mice. In order to examine our hypothesis, three well-investigated antioxidant phytochemicals, curcumin, hesperidin and rutin, were combined into a mixture (CHR) for this study. Diabetes was induced in 6-month-old female ICR mice by STZ (100 mg/kg, i.p.) administration, and CHR or vehicle control was orally administered (200 mg/kg per body weight of each ingredient) to the hyperglycemic mice (blood glucose levels > 250 mg/dl) for a period of 14 days. Administration of CHR to the hyperglycemic mice significantly reduced blood glucose levels, attenuated STZ-induced lipid peroxidation and total nitrate/nitrite levels, and significantly augmented the expression of superoxide dismutase and glutathione in the liver. STZ-induced hyperglycemia resulted in downregulation of antiapoptotic proteins Bcl-2 by 66% and Bcl-XL by 51%, and upregulation of the pro-apoptotic Bad (69%) with an increase in the ratio of cytosolic/mitochondrial cytochrome c by 81% in hepatic tissue. Administration of CHR significantly ameliorated apoptotic signaling in STZ-induced diabetic mice, significantly increasing Bad/Bcl-2 and Bad/Bcl-XL ratios to 410% and 244% respectively in the hyperglycemic group. This study demonstrated that a bioflavonoid mixture of curcumin, hesperidin and rutin (CHR) ameliorates hepatic oxidative stress caused by STZ-induced hyperglycemia, resulting in improved hepatic function and glucose regulation.

Agents of Bioterrorism: Curriculum and Pedagogy in an Online Masters Course.

The Agents of Bioterrorism course (BSBD 640, University of Maryland University College) is a graduate level course created in response to an elevated need for scientists working in the field of medical countermeasures to biological and chemical weapons in the years following 9/11. Students read and evaluate assigned current primary literature articles investigating medical countermeasures at each stage of development. In addition, students learn concepts of risk assessment, comparing and ranking several agents of terror. Student learning is assessed through a variety of assignments. A term paper focuses on a lesser known weapon of terror, with students recommending the best countermeasure in development and delivering a risk assessment comparing their agent to other major weapons of terror discussed throughout the semester. Similarly, a group project on an assigned major weapon of terror (anthrax, plague, smallpox, vesicants, or nerve agent) focuses more heavily on evaluating primary literature and concluding which countermeasure(s) in development are the best. Students complete the course with a fundamental understanding of the mechanism of action of many biological agents, information literacy for the medical literature available at PubMed and the primary scientific literature, and a basic understanding of the role of the government in biodefense research. This paper describes the pedagogical approaches used to teach this course and how they might be adopted for other courses.

Sepiapterin reductase mediates chemical redox cycling in lung epithelial cells.

In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury.

NAD kinase regulates the size of the NADPH pool and insulin secretion in pancreatic ß-cells.

NADPH is an important component of the antioxidant defense system and a proposed mediator in glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells. An increase in the NADPH/NADP(+) ratio has been reported to occur within minutes following the rise in glucose concentration in ß-cells. However, 30 min following the increase in glucose, the total NADPH pool also increases through a mechanism not yet characterized. NAD kinase (NADK) catalyzes the de novo formation of NADP(+) by phosphorylation of NAD(+). NAD kinases have been shown to be essential for redox regulation, oxidative stress defense, and survival in bacteria and yeast. However, studies on NADK in eukaryotic cells are scarce, and the function of this enzyme has not been described in ß-cells. We employed INS-1 832/13 cells, an insulin-secreting rat ß-cell line, and isolated rodent islets to investigate the role of NADK in ß-cell metabolic pathways. Adenoviral-mediated overexpression of NADK resulted in a two- to threefold increase in the total NADPH pool and NADPH/NADP(+) ratio, suggesting that NADP(+) formed by the NADK-catalyzed reaction is rapidly reduced to NADPH via cytosolic reductases. This increase in the NADPH pool was accompanied by an increase in GSIS in NADK-overexpressing cells. Furthermore, NADK overexpression protected ß-cells against oxidative damage by the redox cycling agent menadione and reversed menadione-mediated inhibition of GSIS. Knockdown of NADK via shRNA exerted the opposite effect on all these parameters. These data suggest that NADK kinase regulates intracellular redox and affects insulin secretion and oxidative defense in the ß-cell.

The level of menadione redox-cycling in pancreatic ß-cells is proportional to the glucose concentration: role of NADH and consequences for insulin secretion.

Pancreatic ß-cells release insulin in response to elevation of glucose from basal (4-7mM) to stimulatory (8-16mM) levels. Metabolism of glucose by the ß-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H(2)O(2)), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H(2)O(2) inhibit insulin secretion. Menadione, which produces H(2)O(2) via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on ß-cell metabolism and insulin secretion in INS-1 832/13, a rat ß-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H(2)O(2) production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1-10µM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H(2)O(2) formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H(2)O(2) and menadione on insulin secretion.

Plasma membrane electron transport in pancreatic ß-cells is mediated in part by NQO1.

Plasma membrane electron transport (PMET), a cytosolic/plasma membrane analog of mitochondrial electron transport, is a ubiquitous system of cytosolic and plasma membrane oxidoreductases that oxidizes cytosolic NADH and NADPH and passes electrons to extracellular targets. While PMET has been shown to play an important role in a variety of cell types, no studies exist to evaluate its function in insulin-secreting cells. Here we demonstrate the presence of robust PMET activity in primary islets and clonal ß-cells, as assessed by the reduction of the plasma membrane-impermeable dyes WST-1 and ferricyanide. Because the degree of metabolic function of ß-cells (reflected by the level of insulin output) increases in a glucose-dependent manner between 4 and 10 mM glucose, PMET was evaluated under these conditions. PMET activity was present at 4 mM glucose and was further stimulated at 10 mM glucose. PMET activity at 10 mM glucose was inhibited by the application of the flavoprotein inhibitor diphenylene iodonium and various antioxidants. Overexpression of cytosolic NAD(P)H-quinone oxidoreductase (NQO1) increased PMET activity in the presence of 10 mM glucose while inhibition of NQO1 by its inhibitor dicoumarol abolished this activity. Mitochondrial inhibitors rotenone, antimycin A, and potassium cyanide elevated PMET activity. Regardless of glucose levels, PMET activity was greatly enhanced by the application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle. We propose a model for the role of PMET as a regulator of glycolytic flux and an important component of the metabolic machinery in ß-cells.