SUMMARYElephant seals naturally experience prolonged periods of absolute food and water deprivation (fasting). In humans, rats and mice, prolonged food deprivation activates the renin-angiotensin system (RAS) and increases oxidative damage. In elephant seals, prolonged fasting activates RAS without increasing oxidative damage likely due to an increase in antioxidant defenses. The mechanism leading to the upregulation of antioxidant defenses during prolonged fasting remains elusive. Therefore, we investigated whether prolonged fasting activates the redox-sensitive transcription factor Nrf2, which controls the expression of antioxidant genes, and if such activation is potentially mediated by systemic increases in RAS. Blood and skeletal muscle samples were collected from seals fasting for 1, 3, 5 and 7weeks. Nrf2 activity and nuclear content increased by 76% and 167% at week 7. Plasma angiotensin II (Ang II) and transforming growth factor β (TGF-β) were 5000% and 250% higher at week 7 than at week 1. Phosphorylation of Smad2, an effector of Ang II and TGF signaling, increased by 120% at week 7 and by 84% in response to intravenously infused Ang II. NADPH oxidase 4 (Nox4) mRNA expression, which is controlled by smad proteins, increased 430% at week 7, while Nox4 protein expression, which can activate Nrf2, was 170% higher at week 7 than at week 1. These results demonstrate that prolonged fasting activates Nrf2 in elephant seals and that RAS stimulation can potentially result in increased Nox4 through Smad phosphorylation. The results also suggest that Nox4 is essential to sustain the hormetic adaptive response to oxidative stress in fasting seals.Key words: antioxidants, hormesis, Nox4, oxidative stress, starvation, renin-angiotensin system.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
2871Fasting activates Nrf2 in seals muscle (Vázquez-Medina et al., 2011a;Vázquez-Medina et al., 2011c). Moreover, nuclear accumulation of Nrf2 increases in the skeletal muscle of the elephant seal in response to repetitive bouts of apnea-induced ischemia/reperfusion (Vázquez-Medina et al., 2011c), which are frequent at the end of the post-weaning fast (Thorson and Le Boeuf, 1994).Whether Nrf2 is activated in response to fasting in elephant seals, or any other mammal, has not been investigated. Therefore, the goal of the present study was to elucidate the role of Nrf2 in mediating the adaptive response to oxidative stress during prolonged fasting in a mammal adapted to cope with such conditions, the northern elephant seal. We have previously shown that prolonged fasting increases Nox4 expression in the skeletal muscle of the elephant seal (Vázquez-Medina et al., 2010). Unlike other NADPH oxidases, Nox4 is independent of cytosolic activator subunits, and thus is constitutively active (Martyn et al., 2006;Nisimoto et al., 2010;von Löhneysen et al., 2012). Nox4 is also uniquely localized in several subcellular compartments (Anilkumar et al., 2008; Block et al., 2009;Sun et al., 2011) and produces intracellular hydrogen peroxide (H 2 O 2 ), a p...