Shotgun Proteomics Analysis of Hibernating Arctic Ground Squirrels

Abstract

Mammalian hibernation involves complex mechanisms of metabolic reprogramming and tissue protection. Previous gene expression studies of hibernation have mainly focused on changes at the mRNA level. Large scale proteomics studies on hibernation have lagged behind largely because of the lack of an adequate protein database specific for hibernating species. We constructed a ground squirrel protein database for protein identification and used a label-free shotgun proteomics approach to analyze protein expression throughout the torpor-arousal cycle during hibernation in arctic ground squirrels (Urocitellus parryii). We identified more than 3,000 unique proteins from livers of arctic ground squirrels. Among them, 517 proteins showed significant differential expression comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1-2 months after hibernation had ended (non-hibernating). Consistent with changes at the mRNA level shown in a previous study on the same tissue samples, proteins involved in glycolysis and fatty acid synthesis were significantly underexpressed at the protein level in both late torpid and early aroused animals compared with non-hibernating animals, whereas proteins involved in fatty acid catabolism were significantly overexpressed. On the other hand, when we compared late torpid and early aroused animals, there were discrepancies between mRNA and protein levels for a large number of genes. Proteins involved in protein translation and degradation, mRNA processing, and oxidative phosphorylation were significantly overexpressed in early aroused animals compared with late torpid animals, whereas no significant changes at the mRNA levels between these stages had been observed. Our results suggest that there is substantial post-transcriptional regulation of proteins during torporarousal cycles of hibernation. Molecular & Cellular Proteomics 9:313-326, 2010.Hibernation is a survival strategy of regulated metabolic suppression adopted by a wide range of mammalian species to survive environmental conditions of low or unpredictable food availability (1, 2). Through complex cellular and molecular reorganization, hibernators have evolved the ability to sustain and spontaneously reverse profoundly low levels of body temperature and rates of oxygen consumption. For example, during torpor the arctic ground squirrel, Urocitellus parryii, adopts a core body temperature of Ϫ2.9°C and a minimum metabolic demand that is 2% of its normal, basal metabolic rate (3, 4). However, during the hibernation season, arctic ground squirrels, like all other small mammalian hibernators, alternate between prolonged torpor (to 24 days) and periodic arousal episodes when they spontaneously rewarm to euthermic body temperatures (36 -37°C) for 10 -15 h before returning to torpor. The genetic and molecular mechanisms of regulation and tolerance of hibernation and the functional significance of arousal episodes remain central questions in hibernati...

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