Some creatures are better at coping without oxygen than others. Humans keel over after just three minutes, but goldfish can keep going for months if it's cool enough. Jeff Richards explains that the key to survival is balancing your energy demands. Most creatures that survive low oxygen levels drastically reduce their energy consumption by shutting down processes such as protein synthesis and ion transport to preserve meagre energy supplies. But how do hypoxia-tolerant creatures coordinate the complex array of energy-conserving events that protect them from otherwise certain death? At the time that Richards began puzzling this question, he was working on exercise in muscles. It occurred to him that the challenges of matching energy consumption with supply in exercising muscles were similar to those faced by hypoxia tolerant animals. Knowing that a protein, AMP-activated protein kinase (AMPK), was the key coordinator in exercising muscle, Richards says `a light went on in my head'; maybe AMPK was the master switch for hypoxia tolerance too. He decided to see if AMPK was involved in metabolic regulation in a hypoxia tolerant species: the goldfish ().
Setting up his own lab for the first time, Richards recruited Master's student Lindsay Jibb to begin discovering whether AMPK is significant in hypoxia tolerance. But without a genome to fall back on, Jibb first had to set about cloning individual subunits from the kinase to find out which tissues produce the protein. Not surprisingly, AMPK turned up in the brain and kidney,but the duo also found it in the fish's liver. Knowing that the liver of other hypoxia-tolerant species drastically reduces metabolic activity when faced with low oxygen levels, Richards decided to focus on the role of AMPK in goldfish liver.
Next, the team decided to see how the fish's liver responded to hypoxia. The first thing that they noticed was that ATP levels in the liver dropped significantly during the first 30 min of hypoxia. Richards admits that this was surprising; one of the hallmarks of hypoxia-tolerant species is that their ATP levels remain constant, even when oxygen levels fall. However, after this initial drop, the goldfish's ATP levels stabilised.
Having found that the goldfish were able to maintain their energy supplies,even when oxygen levels were low, Richards and Jibb decided to see if the goldfish's AMPK was capable of activating other proteins. Knowing that AMPK in muscle adds a phosphate molecule to enzymes to activate them, the duo tested whether the goldfish AMPK was also capable of transferring a phosphate molecule to target proteins. Supplying liver tissue containing AMPK with radioactive ATP and a protein fragment that could only be phosphorylated by AMPK, the team found that AMPK successfully transferred a radioactive phosphate from the ATP to the protein fragment. AMPK could active key proteins involved in hypoxia tolerance.
Richards admits that this discovery was very exciting, but then Jibb suggested taking the experiments even further; could goldfish AMPK add a phosphate to a protein that must be switched off to save energy? Could AMPK phosphorylate and inactivate Elongation Factor 2, a key protein in protein synthesis? This time the team used antibodies to identify whether AMPK transferred a phosphate to Elongation Factor 2, and it did. AMPK could switch off protein synthesis.
Richards admits that this is all circumstantial evidence that AMPK is the master regulator of hypoxia tolerance in goldfish, but hopes to prove one day that AMPK is at the hub of hypoxia tolerance.
