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

Abstract

Stensløkken KO, Ellefsen S, Stecyk JAW, Dahl MB, Nilsson GE, Vaage J. Differential regulation of AMP-activated kinase and AKT kinase in response to oxygen availability in crucian carp (Carassius carassius). Am J Physiol Regul Integr Comp Physiol 295: R1803-R1814, 2008. First published October 15, 2008 doi:10.1152/ajpregu.90590.2008.-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 O 2/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 ␣ 2, ␤1B, ␥2a, whereas a more even expression of subunits was found in the brain. In the heart, expression of the regulatory ␥ 2-subunit increased in the heart during anoxia. In the brain, expression of the ␣ 1-, ␣2-, and ␥1-subunits decreased with anoxia exposure, but expression of the ␥ 2-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. metabolism; hypoxia; ATP; ADP; ethanol ANOXIA TOLERANCE IN VERTEBRATES is limited to a few species, the most extreme being the crucian carp (Carassius carassius) and the freshwater turtles (genera Trachemys, Chrysemys, and Chelydra). These ectothermic animals can survive for months without oxygen at temperatures near 0°C (43,50). A key to survival in prolonged anoxia is the successful matching of ATP demand to the limited ATP production from anaerobic glycolysis. The anoxia-tolerant species appear to have evolved different strategies to meet this challenge (32). The turtle is strongly metabolically depressed during anoxia, with a drastically suppressed rate of ATP-turnover. Anoxic Carassius also decreases metabolic rate, but to a more moderate extent (62).The anoxic carp also upregulates glycolysis to match ATP supply and demand (33), but has evolved a unique strategy of producing ethanol as the major end product of anaerobic glycolysis (56). Ethanol is released to the surrounding water to avoid severe acidosis (42), and liver glycogen content is likely...

Effects of environmental hypoxia on cardiac energy metabolism and performance in tilapia

“…Activation of AMPK has recently been implicated in reducing ATP demand of certain tissues as well as whole animal metabolic rate depression in hypoxia-exposed goldfish and crucian carp (28,52). Our results do not suggest a role for AMPK in hypoxia tolerance of tilapia heart (Table 5), and the absence of an increase in [AMP free ] (Table 3) is consistent with the lack of AMPK activation.…”

“…These effects, which are well characterized in mammalian ischemic heart models, include lipotoxicity and uncoupling of glycolysis from glucose oxidation that exacerbates metabolic acidosis and disturbs ion homeostasis (16). Interestingly, this latter event is mediated by AMPactivated protein kinase (AMPK), which is potentially important in decreasing Ṁ O 2 and ATP demand in certain tissues in goldfish (Carassius auratus) and crucian carp during oxygen lack (28,52).…”

Activation and Signaling by the AMP-Activated Protein Kinase in Endothelial Cells

“…For example, under hypoxic but not normoxic conditions, a dominant-negative AMPK mutant abolished the VEGFinduced, Akt-mediated phosphorylation of eNOS, as well as the VEGF-induced tube formation in Matrigel. 84 A similar phenomenon was observed in vivo in carp in response to anoxia, 86 and studies addressing the response to laminar flow in cultured endothelial cells also indicate that the AMPK can negatively regulate Akt, resulting in the transient activation of p70S6 kinase. 54 On the other hand, a dominant-negative AMPK mutant was reported to inhibit Akt phosphorylation in adiponectin-treated endothelial cells, although a dominantnegative Akt mutant failed to affect AMPK activation, allowing the authors to conclude that that the activation of the AMPK must lie upstream from that of Akt.…”

Stellate and pyramidal neurons in goldfish telencephalon respond differently to anoxia and GABA receptor inhibition

“…With anoxia or a shortage of ATP, certain intracellular signaling proteins, such as AMPdependent protein kinase (AMPK), are activated due to an increase in [AMP] (Carling, 2004). AMP has a critical role in maintaining cellular and systemic energy balance and promoting glycolysis (Stensløkken et al, 2008). Through phosphorylation, AMPK strongly enhances GABA B receptor activity, allowing a greater K + efflux via GIRKs (Kuramoto et al, 2007).…”

“…Through phosphorylation, AMPK strongly enhances GABA B receptor activity, allowing a greater K + efflux via GIRKs (Kuramoto et al, 2007). Anoxic exposure significantly increases AMPK phosphorylation in crucian carp brain (Stensløkken et al, 2008).…”