1. NQO1, NAD(P)H dependent cytosolic oxidoreductase, modulates redox status and H2O2 levels in pancreatic -cells Delaine M. Zayas-Bazn Burgos1 and Joshua Gray2, Emma Heart3 1University of Puerto Rico at Cayey; 2US Coast Guard Academy, New London, CT; 3MarineBiological Laboratory, Woods Hole, MA Abstract: Diabetes is the illness in which either the -cells do not secrete enough insulin or secrete insulin that is not efficient. Type 2 diabetes is marked by a reduction in the ability of beta cells of the pancreas to secrete insulin. Redox status, defined as the ratio of the reduced-to-oxidized forms of redox couples (such as NADH-to-NAD+ and NADPH-to-NADP+), plays an important role in overall cell health and in the in glucose-stimulated insulin secretion (GSIS) from pancreatic -cells. Here we have investigated the role of the cytosolic NAD(P)H-dependent oxidoreductase NQO1 on -cell redox status and quinone-dependent production of reactive oxygen intermediates (ROI). In both clonal insulin secreting -cells (INS-1 832/13) and isolated rodent islets, NQO1 over-expression blunted quinone-dependent ROI production, while islets from NQO1 knockout mice had enhanced ROI formation. Furthermore, NQO1 has been found to decrease NAD(P)H-to-NAD(P)+ ratio, consistent with the NQO1-dependent utilization of NAD(P)H for the intrinsic Plasma Membrane Electron Transport activity in -cells. Together, these data show that NQO1 plays an important role in maintaining proper redox status and maintains insulin secretion in the face of oxidative stress. endocrine gland that, in most species, arisesIntroductionfrom ventral and dorsal buds whichBackgroundsubsequently merge to form the pancreas. ThisJdefinition has been conserved and furtherorgen Jonsson and his team indetailed to give everyone a better1994 stated that: Theunderstanding of the pancreas. The pancreasmammalian pancreas is a mixed exocrine andserves both digestive and endocrine functions

2. and it is composed of two main parts. The firstAll of these cells can be directly orpart is known as the Pancreatic Acini andindirectly linked to many of the metabolismcorresponds to the exocrine area of thediseases we know. Metabolism can be definedpancreas. The second main part and our area ofas the essential chemical processes involved ininterest is the endocrine area known as theconverting nutrients into chemical energy andIslets of Langerhans. The Islets of Langerhansmolecularare cluster of cells that produce variousmaintaining the living system. (Hickman 2007).hormones. There are five types of cellsThese chemical processes include digestion,composing the Islets of Langerhans and theiracquisition of energy, respiration and synthesistype dictate the type of hormone they produce,of molecules and structures. Diabetes mellitus,release and under what stimuli. The cells andexocrinetheir function are illustrated in the followingpseudocyst among others are diseases thattable.affect the metabolism by affecting one orcomponentspancreaticforbuildinginsufficiencyandandvarious steps of the digestion and nutrientTable 1.1 Islet Compositionabsorbance of the organism. Type of CellFunction cellsProduce GlucagonIn 1990 Lind C et al identified NQO1, an NAD(P)H-dependent cytosolic oxidoreductase, was for its ability to reduce cellsProduce Insulin cellsProduce Somatosin (Growthquinones such as menadione. Quinones are a class of organic compound that could eitherhormone-inhibiting Hormone)be exogenous or endogenous. Haefeli in 2011stated the features of quinones: quiones cellsProduce GherlinPP cellsProduce Pancreatic Polypeptidefeature a quinoid conjugated double-bond 3. system, which is responsible for theirthe formation of relatively stable quinolselectrophilic nature(hydroquinones), which undergo less redox cycling due to the greater stability of the hydroquinone versus the semiquinone and further detoxification via phase 2 metabolismThis concept is consistent with the increased susceptibility of the NQO1 knockout model mouse to high and toxic doses of redox cycling compounds and xenobiotics evidenced in 2000 by Joseph and his team. This supportsFigure 1 NQO1 reduces H2O2 production by quinones. Quinones such as menadione (1) are reduced by NAD(P)H-dependent oxidoreductase enzymes to either quinols (2) or semiquinols (3).the protective role of NQO1 against oxidative stress in a variety of tissues. However, there areSemiquinols, generated by 1-electron reducation of quinones, are highly reactive and reduce molecular oxygen to generate reactive oxygen intermediates (ROIs), while regenerating the parent quinoneno studies on the protective role of NQO1 in pancreatic islet -cells, which otherwisecompound (1). Complete 2-electron to the quinol (2), however, facilitiates further metabolism and elimination of the quinone. NQO1 catalyzes complete 2-electron reduction of quinones to relatively stablecontain relatively low levels of classical antioxidant enzymes (Tiedge M et al 1997).quinols, which results in a lower level of ROI production.Inside the cell, quinones undergo either one- or two electron NAD(P)H-dependent reduction: 1-electron reduction leads to the formation of unstable semiquinones, which can readily reduce molecular oxygen to superoxide, regenerating the parent quinone compound via redox cycling (Fig. 2). In contrast, complete 2 electron reductions mediated by NQO1 lead toIn parallel to its role as a detoxification enzyme, NQO1 has more recently been implicated in the regulation of intermediary metabolism. NQO1 expression is correlated with fasting insulin levels evidenced in Palming J et al work in 2007 and an association between reduced activity of NQO1 due to polymorphism and dysregulated blood 4. glucose levels has been reported in humans832/13 cells as well as primary rodent islets, we(Kim 2009). Gaiwkad et al evidenced in 2001have demonstrated that NQO1 regulates theNQO1s role in cellular metabolism withlevel of the NAD(P)H/NAD(P)+ ratio, whichstudies using global NQO1 knock-out mice,can explain its effect on the GSIS. Furthermore,which found that these mice suffer from severalusing over-expression or knockout strategies,metabolic defects, including insulin resistancewe have demonstrated that NQO1 reduces thein the periphery, and fail to increase insulindegree of quinone-dependent redox cycling andoutput in the face of their insulin resistance insuperoxide production in -cells, thus acting tocontrast to the normal -cell compensatoryprevent toxicity under enhanced pro-oxidanthyperinsulinemic response (Pi 2007). We haveload. We believe that NQO1 reduces oxidativepreviously demonstrated that NQO1 regulatesstress in -cells by lowering quinone-dependentglucose-stimulated insulin secretion (GSIS) andH2O2 production, through the complete twoPlasma Membrane Electron Transport (PMET)electron reduction. We also believe that at theactivity in pancreatic -cells and our labmetabolic level NQO1 modulates NAD(P)H-continues to investigate the mechanisms of thisto-NAD(P)+ ratio in -cells, as it does in otherpathway.tissues.Problem and hypothesesMaterials and MethodsIn this study we investigated the role ofIn order to conduct the experiments theNQO1 in -cell metabolic pathways, namelyfirst procedures were to over express NQO1 inthe role of NQO1 in modifying the redoxour cell line. This was achieved through thestatus. This is one of the key determinants ofinfection with the adenovirus that either was anthe -cell health and a coupling factor inempty vector, control, or the over expressingglucose-stimulated insulin secretion (GSIS).gene. Then the NQO1 activity was measured.Using both clonal insulin secreting INS-1Whole cell lysates were prepared by sonication 5. followed by centrifugation at 12 kg for 5 min atThen the level of released hydrogen peroxide4 C. Equal concentrations of cell lysate(H2O2) was quantified using Amplexprotein were tested for NQO1 activity, whichRed/horseradish peroxidase. Fluorescence (540was quantified by the decrease in absorbance ofexcitation, 595 emission) was monitored usingdichlorophenolindophenol (DPI) (600 nm) overa SpectraMax M5 multi-mode microplatea period of one minute. The difference inreader (Molecular Devices, Sunnyvale, CA).activity in the absence and presence ofThis was evaluated with both rodent islets anddicoumarol (20 M) are expressed as NQO1INS-1 832/13 cells. The rodent islets wereactivity. Figure 2isolated from normal mice and the global knock out utilizing the corresponding procedures.NQO1 over-expression in INS-1 832/13 cells. Adenoviral-mediated over-expression resulted in the increase of NQO1 protein (adaptedResultsfrom[10]) and enzyme activity, measured as the The effects of NQO1 over-expressionreduction of DCPIP (dichlorophenolindophenol)on menadione-dependent hydrogen peroxide production in INS-1 832/13 cells were measured and analyzed (Figure 3). Dicoumarol (DIC), an inhibitor of NQO1, blocks NQO1ControlNQO1+inhibitory action on redox cycling and H2O2 production. On the first column with no addition of Dicoumarol NQO1s protection can be clearly seen. Under high glucose conditions NQO1 lowers statistically hydrogen peroxide production. Legend: 3G is 3 mM glucose, 16 6. is 16 mM glucose. Data are means SE from 2NQO1 KO. The levels of hydrogen productionexperiments performed in quadruplicateare clearly lowered by the presence of NQO1.measurement.*P 0.05 Control vs. NQO1+ . Figure 3. Effects of NQO1 on H2O2 production in INS-1Figure 4. Effects of NQO1 on H2O2 production in rodent islets832/13 cellsThe effects of NQO1 over-expression (panel A) and knock-down (panel B) on menadionedependent H2O2 production in isolated rodent islets (Figure 4). The over-expressing islets came from normal mice and after isolation were infected with adenovirus. The NQO1 knock-out islets came from the global knockout mice. Data are means SE from 2Lastly in order to evidence the effects ofexperiments performed in quadruplicateNQO1 in the -cells metabolism the NAD(H)measurement. *P 0.05 Control vs. NQO1+ orto- NAD+ ratio was quantified. NQO1 regulates NADH-to-NAD+ ratio in INS-1 7. 832/13 cells and islets. INS-1 832/13 (A),Conclusionsinfected with control adenovirus (Ad-control) or NQO1 over-expressing adenovirus (Ad-All of these results lead to the various conclusions about NQO1 role in -cell healthNQO1) or isolated NQO1 Wild Type and and metabolism. In terms of the health of the NQO1 Knock-Out islets (B) were exposed to cell NQO1 protects from oxidant stress, 4mM or 16 mM glucose and NADH and NAD+ therefore enhances -cell health. In terms of were measured by LC/MS/MS. Data are means metabolism NQO1 modulates -cell redox SE from 2-3 experiments performed in status by lowering the NADH-to-NAD+ ratio as duplicate measurement.*P 0.05 Ad-control vs. it does in other tissues such as liver and Ad-NQO1, and KO vs. WT. adipose. This effect on redox cycle has a direct Figure 5. NQO1 mediates redox cylcingincreasing role in glucose metabolism and glucose stimulated insulin secretion. Future experiments may include exploring NQO1s role in the physiological state. Under normal conditions almost every eukaryotic cell posses the endogenous quinine ubiquinone. Another great follow-up would be to determine the role of NOQ1 under glucotoxicity. Glucotoxicity is high glucose and high fatty acids. 8. AcknowledgementsPalming J et al (2007) J Clin Endocrinol Metab 92: 2346-52This project was conducted thanks toPi J et al (2007) Diabetes 56, 1783-91.the collaboration and help of many individuals. Tiedge M et al (1997) Diabetes 46: 1733-42This work was supported by the National Science Foundation through the Biological Discovery in Woods Hole DBI 1005378, the American Diabetes Association 7-12-BS-073 and National Institute of Health through both Dr. Emma Hearts grant IH R56DK088093, and through the Research Initiative for Scientific Enhancement Program in the University of Puerto Rico at Cayey R25 GM059429. We extend our gratitude towards the REU directors Dr. Mensinger and Dr. Malchow. References Gaikwad A et al (2001) JBC 276: 22559-64 Gray JP et al (2011) AJP Endo Metab 301: E113021. Haefeli RH (2011) PLos One 6: e17963. Joseph P et al (2000) Biochem Pharmacol 60: 207-14 Kim D (2009) Korean Diabetes J 33: 24-30 Lind C et al (1990) Method Enzymol, 186:287-301.