2017 Cardiovascular Research Day Abstract Book

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Regulation of Akt Signaling by NQO1
Joshua Preston 1 • Andrea Di Francesco, PhD 2 • Krystle Kalafut 2 • Tyler Rhinesmith 2 • Clara Di
Germanio, PhD 2 • Michel Bernier, PhD 2 • Rafael de Cabo, PhD 2
1Pharmacology and Nutritional Sciences, University of Kentucky • 2 Translational Gerontology
Branch, National Institute on Aging, National Institutes of Health
Undergraduate
Background: NQO1 is an inducible quinone reductase that participates in the cellular defense
system in response to chemical and oxidative stress. By using NAD(P)H as electron donor, NQO1
enzymatic activity shifts the cellular redox state leading to a significant increase in the NAD+/NADH
ratio. The propensity of NQO1 knockout mice to exhibit a diabetes-like phenotype and a null NQO1
polymorphism associated with metabolic syndrome phenotypes in humans suggests a role for this
enzyme in cellular energetics and metabolism. Here, we explored a possible link between NQO1, an
intracellular generator of NAD+, and nutrient-sensitive NAD+-dependent deacetylase activity in the
regulation of Akt, which plays a central role in regulating cellular signaling and energy pathways
critically involved in type 2 diabetes and associated metabolic disorders.
Results: Pharmacological inhibition of NQO1 with the mechanism-based inhibitor Mac609
increased insulin-stimulated Akt phosphorylation in HepG2 and HeLa cell lines. Consistent with
increased Akt activation, Mac609 promoted nuclear exclusion and inactivation of the downstream
Akt targets, the gluconeogenic transcription factors forkhead box O1 (FOXO1) and O3 (FOXO3a) in
liver-derived HepG2 cells treated with insulin. Conversely, siRNA-mediated NQO1 knockdown
increased insulin- or serum-stimulated Akt phosphorylation. Similarly, MDA-MB-468 cells, which
carry a polymorphic form of NQO1 that results in an absence of NQO1 protein and activity, showed
markedly higher constitutive and insulin-stimulated Akt phosphorylation levels compared to an
isogenic cell line stably expressing NQO1. We then explored the impact of pharmacological
inhibition or genetic down-regulation of NQO1 on the global pattern of protein acetylation in HeLa
cells. The reduction in NQO1 level and/or activity was associated with an increase in protein
acetylation levels likely due to the inhibition of NAD+-dependent sirtuins. Lastly, cell treatment
with the NAD+ precursor nicotinamide mononucleotide (NMN) inhibited Akt phosphorylation in a
dose- and time-dependent manner, thus recapitulating the effects of NQO1 on Akt activity.
Conclusions: We have identified a molecular mechanism whereby NQO1 functions as a modulator
of insulin/Akt signaling pathway. These findings have implications for future basic and
translational research on pathways that control energy homeostasis and diabetes.
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