Differential regulation of AMP-activated kinase and AKT kinase in response to oxygen availability in crucian carp (Carassius carassius)

Abstract

We investigated whether two kinases critical for survival during periods of energy deficiency in anoxia-intolerant mammalian species, AMP-activated kinase (AMPK), and protein kinase B (AKT), are equally important for hypoxic/anoxic survival in the extremely anoxia-tolerant crucian carp ( Carassius carassius). We report that phosphorylation of AMPK and AKT in heart and brain showed small changes after 10 days of severe hypoxia (0.3 mg O2/l at 9°C). In contrast, anoxia exposure (0.01 mg O2/l at 8°C) substantially increased AMPK phosphorylation but decreased AKT phosphorylation in carp heart and brain, indicating activation of AMPK and deactivation of AKT. In agreement, blocking the activity of AMPK in anoxic fish in vivo with 20 mg/kg Compound C resulted in an elevated metabolic rate (as indicated by increased ethanol production) and tended to reduce energy charge. This is the first in vivo experiment with Compound C in a nonmammalian vertebrate, and it appears that AMPK plays a role in mediating anoxic metabolic depression in crucian carp. Real-time RT-PCR analysis of the investigated AMPK subunit revealed that the most likely composition of subunits in the carp heart is α2, β1B, γ2a, whereas a more even expression of subunits was found in the brain. In the heart, expression of the regulatory γ2-subunit increased in the heart during anoxia. In the brain, expression of the α1-, α2-, and γ1-subunits decreased with anoxia exposure, but expression of the γ2-subunit remained constant. Combined, our findings suggest that AMPK and AKT may play important, but opposing roles for hypoxic/anoxic survival in the anoxia-tolerant crucian carp.