Mammalian hibernators are unique in their ability to cope with freezing temperatures. Some mammals can lower their body temperatures to as low as-2.9°C for several weeks. In the golden-mantle ground squirrel, Spermophilus lateralis, this period of low body temperature, called torpor, is interrupted by arousals during which body temperature rises as much as 30°C in a matter of hours. Squirrels cannot survive frosty winters without these arousals. But hibernation researchers know very little about the proteins expressed during arousals that allow squirrels to re-enter torpor after arousals. To investigate the physiology of hibernation, Elaine Epperson and colleagues from the University of Colorado turned to the differential expression of hundreds of proteins, the proteome, or the squirrels'`hibernome'.
Epperson's team separated liver proteins by isoelectric point and by molecular mass. They obtained about 900 spots and estimate that these only represent 3-5% of the squirrels' liver proteins. When they compared summer squirrels with winter squirrels that had completed arousal and were re-entering torpor, they found differences in 84 spots. The team concluded that these proteins were being differentially expressed during arousal in winter squirrels. But here the team encountered a problem: how to identify these spots without a squirrel genome sequence. The team managed to identify 68 of the 84 spots by matching short stretches of amino acids found in the squirrels to a mammalian database that included the rat and mouse genomes,which share many homologous proteins with the distantly related squirrels.
What insight does this natural history perspective of proteomic changes during hibernation offer? At first sight, it confirms what is already known. For example, some of the 68 proteins that the team found were being upregulated during arousal are involved in protein degradation and synthesis. The team also found that several glycogen-producing proteins were upregulated,confirming that glycogen stores are used during arousal and have to be regenerated. Hibernating winter squirrels are known to rely heavily on lipids as a fuel source. Typical for animals relying on lipids as metabolic fuel, the team observed an increase in expression of a fatty-acid-binding protein and a key ketone body-forming enzyme.
Epperson's proteomic approach also reveals interesting novel findings concerning differential expression among members of a protein family. For example, all but one of several enzymes involved in the detoxification of aldehydes resulting from lipid peroxidation were upregulated in winter compared with summer squirrels, indicating that there is an increase in lipid peroxidation during torpor despite fasting. Interestingly, the only enzyme that was downregulated prefers dietary substrates that occur in low abundance during fasting. Members of an esterase protein family, enzymes that hydrolyze fatty acid esters, were either up- or down-regulated in winter squirrels depending on their affinity for fatty acyl esters, indicating subtle and important metabolic differences between summer and winter squirrels. Thus, a proteomic approach can differentiate among several members of a protein family and reveal a great number of differentially expressed proteins.
The extent to which squirrels change the expression of enzymes that regulate major metabolic pathways during arousals probably came as a surprise to the authors. Having found so many interesting alterations in protein expression in just 3% of the squirrels' liver proteins, they are now eager to tap into the vast remaining pool of differentially expressed proteins.