Think you can survive anoxia? Don't hold your breath! Without oxygen, your cells – like those in most vertebrates – are unable to produce enough energy to supply even basic cellular demands and they quickly die. But certain members of the Carassius genus, such as crucian carp, are able to withstand long periods with little to no oxygen at low temperatures, conditions they encounter during winter beneath frozen lakes. Their remarkable anoxia tolerance is thanks to a unique metabolic pathway in their muscle cells that allows pyruvate, an intermediate breakdown product of glucose, to be processed by the cells in a way that still produces energy but does not require oxygen. This anaerobic energy-producing process involves the conversion of pyruvate into ethanol, and this is something that other vertebrates cannot do. However, despite the passage of nearly 4 decades since the discovery of ethanol production by Carassius muscle cells, a team of researchers headed by Norwegian scientists Göran Nilsson and Stian Ellefsen has only just elucidated the molecular mechanisms underlying this metabolic novelty for extreme anoxia tolerance.
When they started their research, the team knew that in the presence of oxygen, pyruvate enters the aerobic metabolic pathway after being converted in a series of steps to acetyl-CoA by the pyruvate dehydrogenase enzyme (PDH) complex. But in the absence of oxygen, Carassius muscle cells instead convert pyruvate into acetaldehyde, which is then converted to ethanol by another enzyme. The researchers wanted to figure out how pyruvate is converted into acetaldehyde, so they began by sequencing every gene component of the PDH complex in Carassius. They soon realized that Carassius are endowed with extra copies of the PDH complex enzymes thanks to a genome duplication event in Carassius ancestors long ago. The team also noticed that some of these extra copies, in particular the first enzymes in the complex (E1α and E1β), which are responsible for initiating pyruvate conversion, now contained important sequence differences that would change their function. Importantly, these sequence modifications were not present in the anoxia-intolerant Carassius cousin the common carp, which further implicated these new genes in bestowing anoxia tolerance.
To test whether the new E1α and E1β were responsible for driving pyruvate towards ethanol synthesis under anoxic conditions, the research team kept Carassius in either fully oxygenated water or oxygen-depleted water for up to 1 week and then quantified and compared the expressions of the old and newly identified E1α and E1β genes. As suspected, the researchers found that Carassius muscle, the major ethanol-producing tissue during anoxia, expressed considerably more of the new E1α and E1β genes than the old ones (about 100 times more). They also found that brain, which does not produce ethanol during anoxia, only expressed the old E1α and E1β genes. Interestingly, the team showed that this expression pattern was the same irrespective of the oxygen content in the fish's aquaria; instead, anoxia changes the phosphorylation state of E1α and E1β in Carassius muscle. As regulating phosphorylation takes a fraction of the time that it takes to transcribe and translate new proteins from genes, this means that Carassius are primed for anoxia and can rapidly switch between the aerobic and anaerobic pyruvate pathways.
Now you can breathe easy – the mystery of ethanol production in Carassius muscle cells is solved!