NQO1 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 is coupled to insulin secretion in pancreatic β-cell

The role of NQO1 in metabolism

Detoxification enzyme NQO1 knock-out mice

are sensitive to oxidant stress Joseph P et al (2007), Radjendirane V. (1998)

Intermediary metabolism NQO1 levels correlated

with fasting insulin levels Palming J et al (2007)

NQO1 knock-out mice have metabolic defects Gaikwad A et al (20o1)

NQO1 is cytosolic NAD(P)H-dependent Quinone Oxidoreductase 1

Quinones are? Class of organic compound

Exogenous Food: Thymoquinone Pollution: Athraquinone

Endogenous Ubiquinone (CoQ10) Electron carrier Mitochondrial: known function All membranes including Plasma membrane

▫PMET: Plasma Membrane Electron Transport

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

Why?Oxidants are important for signaling in β-

cell under physiological situationPathological high doses of oxidants will

harm β-cell to greater extent than other cell types

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

Mediated by NQO1

Is the transfer of electrons (reduced equivalents)out of the cell: PMET is plasma membrane analog of the mitochondrial ETC

NQO1 is a member of PMET(Plasma Membrane Electron Transport)

Gray et al (2011)

Hypotheses to test:

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 measured

in cell lysate by decrease in absorbance of DCPIP (dichlorophenolindophenol)

▫Activity increased >10x

NQO1+

contro

l

NQO1 (31KD)

Western blot of NQO1 protein in lysates from INS-1 832/13 cells (adapted from Gray et al 2011)

3.3E-10 ± 5.16E -12 5.6E-9 ± 1.96E-12

mol 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

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. • NADH and NADPH are substrates for NQO1• Gaikwad et al (2001): NQO1 has been show

to modulate redox ratios in fat and liver

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