Proteomic analysis of the winter-protected phenotype of hibernating ground squirrel intestine

Abstract

The intestine of hibernating ground squirrels is protected against damage by ischemia-reperfusion (I/R) injury. This resistance does not depend on the low body temperature of torpor; rather, it is exhibited during natural interbout arousals that periodically return hibernating animals to euthermia. Here we use fluorescence two-dimensional difference gel electrophoresis (DIGE) to identify protein spot differences in intestines of 13-lined ground squirrels in the sensitive and protected phases of the circannual hibernation cycle, comparing sham-treated control animals with those exposed to I/R. Protein spot differences distinguished the sham-treated summer and hibernating samples, as well as the response to I/R between summer and hibernating intestines. The majority of protein changes among these groups were attributed to a seasonal difference between summer and winter hibernators. Many of the protein spots that differed were unambiguously identified by high-pressure liquid chromatography followed by tandem mass spectrometry of their constituent peptides. Western blot analysis confirmed significant upregulation for three of the proteins, albumin, apolipoprotein A-I, and ubiquitin hydrolase L1, that were identified in the DIGE analysis as increased in sham-treated hibernating squirrels compared with sham-treated summer squirrels. This study identifies several candidate proteins that may contribute to hibernation-induced protection of the gut during natural torpor-arousal cycles and experimental I/R injury. It also reveals the importance of enterocyte maturation in defining the hibernating gut proteome and the role of changing cell populations for the differences between sham and I/R-treated summer animals.

Adaptive mechanisms regulate preferred utilization of ketones in the heart and brain of a hibernating mammal during arousal from torpor

“…1). Higher levels of BHB in ground squirrel liver (28), as well as an increase in the level of the ketone producing enzyme hydroxy methyl glutaryl Coenzyme A synthase 2 in liver (6) and intestine (18), indicates heightened ketone production during hibernation. Adaptive mechanisms for BHB utilization during hibernation include induction of the BHB transporter MCT1 in the brain (Fig.…”

Multistate proteomics analysis reveals novel strategies used by a hibernator to precondition the heart and conserve ATP for winter heterothermy

“…DiGE labeling was completed as previously described (46). A pooled reference standard containing equal amounts (g) of n ϭ 3 heart protein extracts from each sampling group (24 samples total) was stored in single-use aliquots.…”

“…Spots were considered unambiguously identified if they returned an overall score Ն30 and contained Ն2 peptides for only one protein. If multiple proteins were identified in a spot but one (with Ն2 peptides) had 4ϫ greater spectral intensity than any other, this was also accepted as the unique ID, as previously described (46). The resulting peptide data are available in Supplemental Table S1.…”

Renal adaptation during hibernation

“…As a further complication in the nomenclatures used, some investigators examine monthly or seasonal changes whereas others examine changes that occur during one torpor-arousal cycle of a few days duration. More recent studies have employed specific definitions of stages, representing both seasonal and torpor-arousal cycles, based on CBT monitoring using radiotelemetry (62,63,70,83) (Fig. 1).…”