NQO1, NAD(P)H dependent cytosolic oxidoreductase, modulates redox status and H2O2 levels in pancreatic β-cells

Delaine Zayas-Bazán Burgos1 , Joshua Gray2,3, Emma Heart3

1University of Puerto Rico at Cayey2U.S. Coast Guard Academy3Marine Biology Laboratory

Pancreas: Islets of Langerhans

http://discovery.lifemapsc.com/in-vivo-development/pancreas/pancreatic-ducts

insulin

Glucose Metabolism

The role of NQO1 in metabolism

Detoxification enzyme

Intermediary metabolism Correlated with fasting

insulin levels Palming J et al (2007)

NQO1 knock-out mice Gaikwad A et al (2001)

NQO1 and Quinones

NQO1 knock-out mice have an enhanced sensitivity to menadione suggesting that the principal function is xenobiotic quinone detoxification. Radjendirane V. (1998)

Quinones are? Class of organic

compound Endogenous

Ubiquinone (CoQ10) Electron carrier Mitochondrial: well

known function Exogenous

Food: Thymoquinone Pollution: Athraquinone

Hydroquinone form

Semi-quinone form: reactive

NQO1 reduces H2O2 production by quinones: might be especially important for β-cells, since they have a relatively low level of classical antioxidant enzymes.

NQO1 metabolizes Quinones

NQOI cytoprotection in β-cells

β-cells have relatively low levels of classical antioxidant enzymes SOD • Superoxide dismutases 1,2 and 3

Oxidant signalingUnder stress

Backfires

O

O

O

O

O

O

OH

OH

NAD(P)H NAD(P)+

ubiquinone ubiquinonol

O2O2

extracellular space

cytosolnet

ele

ctro

n f

low

oxidoreductases

NQO1

Is the transfer of electrons

Mediated by NQO1

NQO1 is a member of PMET Gray et al (2011)

Problem and Hypotheses

Objective: to determine role of NQO1 in β-cell health and metabolism.

Hypotheses: (1) NQO1 reduces oxidative stress

in β-cells by lowering quinone-dependent H2O2 production.

(2) NQO1 modulates NAD(P)H-to-NAD(P)+ ratio in β-cells.

Methods

INS-1 832/13 cells

• Over-expression of NQO1▫ Adenovirus with NQO1▫NQO1 activity was

measured in cell lysate by decrease in absorbance of (DPIP) dichlorophenolindophenol activity increased more than 10 fold

• NQ01

+• cont

rol

• NQO1 (31KD)Western blot analysis of NQO1

protein in lysates from INS-1832/13 cells (adapted from Gray et al 2011)

3.3E-10 ± 5.16E -12 5.6E-9 ± 1.96E-12 rmol DCPIP//min

Effect of NQO1 over-expression on menadione-dependent H2O2 production in INS-1 832/13 cells

Effect of NQO1 over-expression and knock-out (KO) on menadione-dependent H2O2 production in isolated

rodent islets

Over-expression Knock-out

Objective: to determine role of NQO1 in β-cell health and metabolism.

Hypotheses: (1) NQO1 reduces oxidative stress in β-

cells by lowering quinone-dependent H2O2 production.

(2) NQO1 modulates NAD(P)H-to-NAD(P)+ ratio in β-cells.

Problem and Hypotheses

NQO1 regulates NADH-to-NAD+ ratio in INS-1 832/13 cells and rodent islets

INS-1 832/13 Rodent Islets

Conclusions

NQO1▫Protects β-cell from oxidant stress▫Modulates β-cell redox status by lowering the NADH-to-NAD+ ratio: effect on metabolism (GSIS)

Future Studies

Role of NQO1 under metabolic stress: high glucose + high fatty acids (glucolipotoxicicty)

Role of NQO1 in PMET

Role of NQO1 in Ubiquinone redox status

Acknowledgments