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ANIMALS THAT SURVIVE WITHOUT
OXYGEN
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19. november 2015
av professor Göran E. Nilsson, Universitetet i Oslo
Ischemic and hypoxic diseases, affecting the brain and heart, are major
causes of death in the industrialized world. The brain and heart are particularly anoxia sensitive, not only in mammals but also in most other vertebrates, including fish (Nilsson et al., 1993; Hylland et al., 1995; Lutz et al.,
2003). Without any oxygen (anoxia), ATP can only be produced by anaerobic
metabolism, a process that yields less than 1/10th of oxidative ATP production.
Due to their high energy demands, the brain and heart are rapidly experiencing falling ATP levels in anoxia. Thus, for most vertebrates, anoxia is
synonymous with death. If deprived of oxygen, brain and heart ATP levels
plummet within minutes, which lead to a stop in ion pumping and, soon, a
general depolarization and loss of ion homeostasis. The resultant drastic
rise in intracellular Ca2+ initiates a deadly cascade leading to necrosis or
apoptosis (Lutz et al., 2003; Garg et al., 2005, for reviews).
Unfortunately, medical science has had a very limited success in counteracting these deleterious effects of ischemia/hypoxia in humans. However, a fresh view on the problem can come from studies on vertebrates that
readily survive hypoxia and even anoxia (Fig. 1). The best studied examples
of such anoxia tolerant animals are some North American freshwater turtles
(genera Trachemys and Chrysemys) and a Scandinavian freshwater fish, the
crucian carp (no: Karuss, lat: Carassius carassius) (Fig. 2). Our two decades
of research on anoxia tolerance has been characterized by a broad approach
to the problem, as this is largely a virgin area of research where many basic
questions need answers. The studies have ranged from finding neurochemical mechanisms for lowering brain energy use, to studies of metabolic and
physiological adjustments of the circulatory and respiratory systems. We
have shown that the crucian carp has a brain that maintains ATP levels and
remain active in anoxia (reviewed by Nilsson, 2001; Nilsson and Lutz,
2004), a heart that can maintain full cardiac output without any oxygen (Ste-
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Fig. 1. Anoxic survival time in “normal” vertebrates and in anoxia tolerant
vertebrates. Note that cold blooded vertebrates in general are as sensitive
to anoxia as mammals, if temperature is taken into account, and that the
anoxia-tolerant vertebrates survive anoxia about 1000 times longer than
other vertebrates. In general, metabolic rate falls sharply with body temperature and a main reason why anoxia tolerant vertebrates survive anoxia
longer at cold temperatures, is that their glycogen stores last longer. For
anoxia-intolerant vertebrates, a main benefit of low temperature is that it
slows down the loss of ATP and the onset of degenerative processes.
cyk et al., 2004), and in its gills, there is a cell mass that undergoes apoptosis in response to hypoxia in a functional and clearly adaptive manner
aimed at increasing the respiratory surface area (reviewed by Nilsson et al.,
2012).
In addition to the difficulty of maintaining ATP production, a major
problem with anoxia is the build up of lactate – the main end-product of
anaerobic glycolysis (Nilsson, 2010, for an overview). The resultant lactic
acidosis is not only painful (as anyone knows that has overreached the aero-
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Fig. 2. The crucian carp (karuss in Norwegian, Carassius carassius in Latin) is
a common inhabitant in many small
lakes and ponds in Northern Europe.
Because of its extreme tolerance to low
oxygen levels, it is often the only fish
species in habitats that become anoxic
due to thick ice and snow coverage
during the winter.
bic capacities of their muscles), but
soon deadly if oxygen is not restored.
In 1958, Blazka found that the crucian
carp differed from other vertebrates
studied by not showing an oxygen debt
after anoxia exposure. Oxygen debt is
the additional need for oxygen uptake
shown by an animal after oxygen supply has been temporarily insufficient,
and is largely caused by the metabolism of lactate.
How the crucian carp avoided an oxygen debt remained a mystery until
1980, when Shoubridge and Hochachka published a paper in Science showing that the goldfish (Carassius auratus), a close relative to the crucian carp,
has the ability to produce ethanol as the major metabolic end product during anoxic conditions, thereby avoiding a build-up of lactate and acidosis.
It was soon shown that also crucian carp produce ethanol in anoxia (Johnston and Bernard, 1983, Nilsson, 1988).
During the following decade several research groups contributed to clarifying some of the underlying mechanisms (Fig. 3). It was found that the ethanol
production only occurred in white and red skeletal muscle, where a very
high activity of alcohol dehydrogenase (ADH) is found (Nilsson, 1988). All
other tissues are virtually devoid of ADH in Carassius (other vertebrates, including humans, have most of the ADH in the liver) so most tissues in these
fishes will produce lactate in anoxia and release this into the blood stream
for transport to the muscle. In the muscle, lactate dehydrogenase catalyzes
the formation of pyruvate from lactate, and when pyruvate enters the pyruvate dehydrogenase complex (PDH), it was suggested that acetaldehyde
somehow leaks out from the complex. This is highly unusual as this enzyme complex shuttles the intermediates and normally only releases the end
product, acetyl-CoA, which is fed into the citric acid cycle. The production
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Fig. 3. The ethanol-producing pathway in crucian carp and goldfish. The
pathway is confined to muscle tissue, while all other organs have to produce lactate in anoxia, which is transported in blood to the muscle. The
ethanol is subsequently released into the blood and leaves the fish by diffusion over the gills. PDH = pyruvate dehydrogenase complex. ADH = alcohol dehydrogenase.
of acetaldehyde from pyruvate is clearly the key step that makes ethanol
production possible, and sets the crucian carp and goldfish aside from other
vertebrates. The acetaldehyde produced is finally transformed by ADH to
ethanol, which due to its lipophilicity diffuses out of the muscle and into the
blood stream and finally leaves the fish through the gills (Fig. 3). We have
suggested that one function of our finding of maintained cardiac output in
anoxic crucian carp is to allow a rapid release of ethanol to the water (Stecyk et al., Science 2004). In other words, the crucian carp probably needs
to pump sufficient blood through the gills to avoid getting alcohol intoxicated during the long anoxic winter.
Obviously, it is high time to utilize modern molecular techniques to better clarify the only ethanol producing pathway known in vertebrates. We
have started on this endeavor, and it should give us insight not only into
how the pathway works, and the genes, proteins, and regulatory mechanisms involved, but also how it evolved. By finding the mutations that made
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this highly unusual pathway functional in the genus Carassius, we aim to
reconstruct its evolution. Our studies show that a genome duplication that
happened in an ancestor to the crucian carp and goldfish a few million years
ago resulted in double copies of the PDH genes. This allowed evolution to
“play” with the extra copies while retaining the “old copies” needed to
maintain the essential function of the PDH complex. The final result is a
new enzyme – the first vertebrate pyruvate decarboxylase, analogous to that
of brewers yeast, and performing the same chemical reaction, allowing the
crucian carp to be its own brewery during the long anoxic winter.
References
Blazka P. (1958): The anaerobic metabolism of fish. Physiological Zoology
31, 117–128.
Garg, S., Narula, J. and Chandrashekhar, Y. (2005): Apoptosis and heart
failure. Journal of Molecular and Cellular Cardiology 38, 73–9.
Hylland, P., Nilsson, G.E. and Johansson, D. (1995): Anoxic brain failure in
an ectothermic vertebrate: release of amino acids and K+ in rainbow
trout thalamus. American Journal of Physiology 269, R1077-R1084.
Hylland, P. and Nilsson, G.E. (1999): Extracellular levels of amino acid
neurotransmitters during anoxia and forced energy deficiency in crucian
carp brain. Brain Reserch 823, 49–58.
Johnston I.A. and Bernard L.M. (1983): Utilization of the ethanol pathway
in carp following exposure to anoxia. Journal of Experimental Biology
104, 73–78.
Lutz, P.L., Nilsson G.E. and Prentice, H. (2003): The Brain Without Oxygen.
3rd Edition. Heidelberg: Springer.
Nilsson, G.E. (1988): A comparative study of aldehyde dehydrogenase and
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sensitivity to anoxia in fish as reflected by changes in extracellular potassium-ion activity. American Journal of Physiology 264, R250-R253.
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Nilsson, G.E. and Lutz. P.L. (2004): Anoxia tolerant brains. Journal of Cerebral Blood Flow and Metabolism 24, 475–486.
Nilsson, G.E., Dymowska, A., Stecyk, J.A.W. (2012): New insights into
the plasticity of gill structure. Respiratory Physiology and Neurobiology
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in vertebrate anaerobic metabolism. Science 209, 308–309.
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