Metabolic response of the Siberian wood frog Rana amurensis to extreme hypoxia

Abstract

the Siberian wood frog Rana amurensis is a recently discovered example of extreme hypoxia tolerance that is able to survive several months without oxygen. We studied metabolomic profiles of heart and liver of R. amurensis exposed to 17 days of extreme hypoxia. Without oxygen, the studied tissues experience considerable stress with a drastic decrease of ATP, phosphocreatine, and NAD+ concentrations, and concomitant increase of AMP, creatine, and NADH. Heart and liver switch to different pathways of glycolysis with differential accumulation of lactate, alanine, succinate, as well as 2,3-butanediol (previously not reported for vertebrates as an end product of glycolysis) and depletion of aspartate. We also observed statistically significant changes in concentrations of certain osmolytes and choline-related compounds. Low succinate/fumarate ratio and high glutathione levels indicate adaptations to reoxygenation stress. Our data suggest that maintenance of the ATP/ADP pool is not required for survival of R. amurensis, in contrast to anoxia-tolerant turtles. Anoxia is a huge stress for the majority of vertebrates. Amphibians are considered to be relatively anoxia-intolerant, in contrast to a few turtle species and certain fish species 1-3 : the most resistant species are known to survive anoxia for a few days at most. However, a recent study 4 proved that the Siberian wood frog Rana amurensis can tolerate almost complete anoxia at 2-3 °C (below 0.2 mg/L oxygen, i.e., less than 1.5% of the normal concentration at this temperature) for several months. This is on par with the best vertebrate models from other classes. Moreover, while red-eared slider turtles, the most studied anoxia-tolerant tetrapods, are dormant under anoxia, the Siberian wood frog is able to react to stimuli, e.g., to flee when disturbed. R. amurensis is thus a unique model of extreme hypoxia tolerance among terrestrial vertebrates. The natural range of R. amurensis includes Northeastern China and Siberia from the Urals to the coast of the Okhotsk sea; its northern distribution is limited by about 71° N. It overwinters under ice in water bodies. Many of these lakes are shallow (< 3 m) due to thermokarst origin, i.e. resulting from local melting of permafrost. Experimental studies 4 demonstrated that during the winter oxygen is depleted in overwintering sites till almost complete anoxia. Siberian wood frogs overwinter for 6 to 7 months in different regions under hypoxia until they can leave the lakes in April-early May. The mechanisms of adaptation to the lack of oxygen are totally unknown for R. amurensis. Here we made an attempt to study this phenomenon using 1 H-NMR-based quantitative metabolomics. This method simultaneously yields concentrations of multiple metabolites 5. At the present, the detailed quantitative metabolomic composition is known for many human tissues, but for very few animals 6-8. To the best of our knowledge, this is the first report on the quantitative metabolomic analysis of amphibian tissues. We determined concentrations of ove...

The Metabolomic Response of Crucian Carp (Carassius carassius) to Anoxia and Reoxygenation Differs between Tissues and Hints at Uncharacterized Survival Strategies

“…Additionally, alanine may be transported to the liver, where it has been suggested to act as a precursor to gluconeogenesis [ 58 , 60 ]. Interestingly, large alanine accumulation in liver tissue has also been seen in the Siberian wood frog Rana amurensis during extreme hypoxia [ 61 ], indicating that alanine may be part of a common mechanism for anoxia-tolerant as well as hypoxia-tolerant species. This additional source of energy would potentially reduce the rate of glycolysis and in this way conserve glucose, which would benefit the crucian carp greatly during prolonged anoxia exposures.…”

Metabolomics improves the histopathological diagnosis of asphyxial deaths: an animal proof-of-concept model

“…This mechanism represents a response to hypoxia based on a coordinate slowdown of both ATP production and consumption to save energy and diminish ROS and other toxic end-products accumulation. Quite recently, the ability to maintain low succinate levels during extreme hypoxic conditions was associated to hypoxia tolerance in amphibians and reptile vertebrates 38 .…”

Biochemical Response to Freezing in the Siberian Salamander Salamandrella keyserlingii

“…This is in apparent contrast to hypoxia in the Siberian wood frog R. amurensis. A study using the same methods [14] revealed reduced ATP/ADP ratio but no products of nucleotide degradation in response to aqueous hypoxia in this species, and the levels of GPC and choline were mostly reduced, not elevated. It looks like that the observed changes are well tolerated, since −8 • C is far from critical for S. keyserlingii.…”

“…In vertebrates, suppression of oxidative phosphorylation is believed to result in the accumulation of high quantities of succinate, which is caused by the reversal of the activity of succinate dehydrogenase in the absence of oxygen [41]. Accumulation of succinate under hypoxia was detected in the Siberian wood frog Rana amurensis [14], the crucian carp Carassius carassius [42], and the red-eared slider turtle Trachemys scripta [43]. Surprisingly, we found no changes in succinate level in the liver, while in the muscle its average concentrations even decrease approximately twofold (Figure 4B).…”

“…The aim of this study was to investigate the biochemical response of the Siberian salamander to freezing and to compare it to that observed in other freeze-tolerant amphibians, as well as to the changes in the metabolome of the hypoxia-tolerant Siberian wood frog R. amurensis. Metabolomic data are currently available only for the latter species [14], enabling direct comparison.…”