When physiologist Markus Frederich decided to shift his research focus from Antarctic crustaceans to mammalian energy metabolism, he didn't anticipate that his disparate research interests would neatly dovetail to lead him into uncharted scientific territory. As a postdoctoral researcher at Harvard Medical School, Frederich was introduced to AMP-activated protein kinase(AMPK), which acts as a metabolic `master switch' to stimulate production of ATP, the cell's main energy source. He explains that while AMPK is well studied in mammals, the existence of this key energy regulator is virtually unexplored in the invertebrate world. From his earlier research, Frederich knew that rising temperatures lead to plummeting energy levels in Antarctic crustaceans. Given that low energy levels trigger AMPK in mammals, he wondered whether heat stress might activate AMPK in crustaceans(p. 722).
To test this, Frederich and his colleagues Michaela O'Rourke, Nathan Furey and Jennifer Jost set out to compare AMPK activity levels in heat-stressed crabs with levels of a traditional heat stress marker, heat shock protein 70(HSP70). A friendly local lobster fisherman supplied rock crabs to Frederich's lab at the University of New England in Maine. The team first investigated how the crabs coped at high temperatures by settling the animals into tanks kept at a comfortable 12°C, then quickly ramped the heat up to 30°C. To identify the first behavioural warning signs of heat stress, they tested the crabs' reaction times at different temperatures by flipping the crabs upside down and timing how long it took for the creatures to right themselves. `Above 18°C, the crabs were noticeably slower than at lower temperatures,suggesting that they were starting to struggle,' says Frederich. The team also assessed the rock crabs' critical temperature – the temperature at which the animals switch to anaerobic metabolism due to heat stress – by measuring the crabs' heart rates and lactate accumulation, and found that it was 26°C.
But would any of these behavioural and physiological findings correlate with molecular markers of heat stress? To find out, the team measured AMPK activity and AMPK and HSP70 protein levels at 2°C increments. Levels of HSP70, the traditional heat stress marker, only started to increase at 28°C – around the crabs' critical temperature. But to Frederich's delight, AMPK activity steadily increased above 18°C, revealing that the crabs were already heat stressed at this lower temperature. Importantly, the threshold indicated by rising AMPK activity corresponded to the animals'behavioural threshold – the temperature at which the crabs struggled to flip themselves over. `Long before crabs reach the temperature at which HSP70 reveals heat stress, other processes indicate that the animals are struggling to cope. AMPK seems to be an earlier, more sensitive heat stress indicator,'says Frederich.
Just to make sure that AMPK and HSP70 produced similar responses in a more traditional prolonged heat stress test, the team also tested crabs sweltering at a continuous 26°C. Sure enough, both HSP70 and AMPK mRNA levels increased constantly over 6 h, showing that rock crabs also have slower but longer-term mechanisms to survive soaring temperatures.
Frederich anticipates potential applications of his team's findings in models to assess how animals will cope with global warming. `AMPK activity could allow us to assess more accurately whether animals are heat stressed,'he concludes.