Project description:Differential gene expression in a wide range of tissues including brown adipose tissue (BAT), liver, heart, hypothalamus, and skeletal muscle in hibernating arctic ground squirrels during multiple stages in torpor-arousal cycles compared to non-hibernating (post-reproductive) animals with illumina beadarray technology. Keywords: Multiple stage comparison
Project description:Hibernating mammals undergo a dramatic drop in temperature and blood flow during torpor and must suppress hemostasis to avoid stasis blood clotting. In addition, cold storage of most mammalian platelets induces cold storage lesions, resulting in rapid clearance following transfusion. 13-lined ground squirrels (Ictidomys tridecemlineatus) provide a model to study hemostasis and cold storage of platelets during hibernation because, even with a body temperature of 4-8C, their platelets are resistant to cold storage lesions. We quantified and systematically compared proteomes of platelets collected from ground squirrels at summer (activity), fall (entrance), and winter (topor) to elucidate how molecular-level changes in platelets may support hemostatic adaptations in torpor. Platelets were isolated from squirrel blood collected in June, October, and January. Platelet lysates from each animal were digested with trypsin prior to 11-plex tandem mass tag (TMT) labeling, followed by LC-MS/MS analysis for relative protein quantification. We found over 700 platelet proteins with significant changes over the course of entrance, torpor, and activity – including systems of proteins regulating translation, platelet degranulation, metabolism, complement, and coagulation cascades. We also noted species specific differences in hemostatic, secretory, and inflammatory regulators in ground squirrel platelets relative to human platelets. In addition to providing the first ever proteomic characterization of platelets from hibernating animals, our results support a model whereby systematic changes in metabolic, hemostatic, and other proteins support physiological adaptations in torpor. In addition, our results could translate into better methods to cold store human platelets, increasing their supply and quality for transfusions.
Project description:Mammalian hibernation is characterized by metabolic rate depression and a strong decrease in core body temperature that together create energy savings such that most species do not have to eat over the winter months. Brown adipose tissue (BAT), a thermogenic tissue that uses uncoupled mitochondrial respiration to generate heat instead of ATP, plays a major role in rewarming from deep torpor. The present study used label-free phosphoproteomics to investigate changes in BAT from thirteen-lined ground squirrels (Ictidomys tridecemlineatus), comparing euthermic squirrels with squirrels in deep torpor. Differential expression of mitochondrial proteins was also investigated. Surprisingly, mitochondrial membrane and matrix protein expression in BAT was largely constant between active euthermic squirrels and their hibernating counterparts. Validation by immunoblotting confirmed that the protein levels of mitochondrial respiratory chain complexes were largely unchanged. However, phosphoproteomics showed that pyruvate dehydrogenase (PDH) phosphorylation increased during ground squirrel hibernation, and this was confirmed by immunoblotting with phospho-specific antibodies. PDH phosphorylation leads to its inactivation, which suggests that BAT carbohydrate oxidation is inhibited during hibernation. Phosphorylation of hormone-sensitive lipase (HSL) also increased during hibernation, suggesting that HSL would be in a partly activated state in BAT to produce the fatty acids that are likely the primary fuel for thermogenesis during arousal.
Project description:Differential gene expression in a wide range of tissues including brown adipose tissue (BAT), liver, heart, hypothalamus, and skeletal muscle in hibernating arctic ground squirrels during multiple stages in torpor-arousal cycles compared to non-hibernating (post-reproductive) animals with illumina beadarray technology. Arctic Ground Squirrels were sampled at four stages of hibernation: early arousal denoted as EA (1-2 hrs after Tb cross 30¡C, n=4), late arousal denoted as LA (7-8 hrs after Tb cross 30¡C, n=4), early torpor denoted as ET (10-20% of torpid episode, n=4) and late torpor denoted as LT (80-90% of torpid episode, n=5), where Tb is the body temperature and the length of torpid episode is estimated from the previous torpor bout. Post-reproductive animals denoted as PR (n=7) were used as non-hibernating control. Five tissue types: brown adipose tissue (BAT), liver, heart, hypothalamus, and skeletal muscle were hybridized on two customized 700-gene beadarray platforms: 1A and 2A on 96-sample Illumina ArrayMatrix. The data of a pilot study involving brown adipose tissue (BAT), liver, and skeletal muscle on 16-sample Illumina BeadChip denoted as 16chip are also included in this series.
Project description:Hibernation is energy saving adaptation involving suppression of activity to survive in highly seasonal environments. Immobility and disuse generate muscle loss in most mammalian species. In contrast to other mammals, bears and ground squirrels demonstrate limited muscle atrophy over the physical inactivity of winter hibernation. This suggests that hibernating mammals have adaptive mechanisms to prevent disuse muscle atrophy. To identify common transcriptional program underlying molecular mechanisms preventing muscle loss, we conducted a large-scale gene expression screening in hind limb muscles comparing hibernating and summer active black bears and arctic ground squirrels by the use of custom 9,600 probe cDNA microarrays. The molecular pathway analysis showed an elevated proportion of overexpressed genes involved in all stages of protein biosynthesis and ribosome biogenesis in muscle of both species during hibernation that implies induction of translation at different hibernation states. The induction of protein biosynthesis likely contributes to attenuation of disuse muscle atrophy through prolonged periods of immobility and starvation. This adaptive mechanism allows hibernating mammals to maintain full musculoskeletal function and preserve mobility during and immediately after hibernation, thus promoting survival. The lack of directional changes in genes of protein catabolic pathways does not support the importance of metabolic suppression for preserving muscle mass during winter. Coordinated reduction of multiply genes involved in oxidation reduction and glucose metabolism detected in both species is consistent with metabolic suppression and lower energy demand in skeletal muscle during inactivity of hibernation. Arctic ground squirrels sampled during winter hibernation were compared with the animals sampled during summer. Muscle was hybridized on a custom 9,600 probes nylon membrane microarray platform. Ten in late torpor, four in early arousal, then in late arousal were studied in experiments.
Project description:Identify shifts in gene expression relevant to torpor phenotypes and recovery following torpor in five tissues of the 13-lined ground squirrel. Sampled tissues and time points overlap with prior hibernation RNA-seq studies in 13-lined ground squirrel and other species, allowing for the analysis of conserved gene expression patterns in torpor.
Project description:Hibernation is energy saving adaptation involving suppression of activity to survive in highly seasonal environments. Immobility and disuse generate muscle loss in most mammalian species. In contrast to other mammals, bears and ground squirrels demonstrate limited muscle atrophy over the physical inactivity of winter hibernation. This suggests that hibernating mammals have adaptive mechanisms to prevent disuse muscle atrophy. To identify common transcriptional program underlying molecular mechanisms preventing muscle loss, we conducted a large-scale gene expression screening in hind limb muscles comparing hibernating and summer active black bears and arctic ground squirrels by the use of custom 9,600 probe cDNA microarrays. The molecular pathway analysis showed an elevated proportion of overexpressed genes involved in all stages of protein biosynthesis and ribosome biogenesis in muscle of both species during hibernation that implies induction of translation at different hibernation states. The induction of protein biosynthesis likely contributes to attenuation of disuse muscle atrophy through prolonged periods of immobility and starvation. This adaptive mechanism allows hibernating mammals to maintain full musculoskeletal function and preserve mobility during and immediately after hibernation, thus promoting survival. The lack of directional changes in genes of protein catabolic pathways does not support the importance of metabolic suppression for preserving muscle mass during winter. Coordinated reduction of multiply genes involved in oxidation reduction and glucose metabolism detected in both species is consistent with metabolic suppression and lower energy demand in skeletal muscle during inactivity of hibernation. Black bears sampled during winter hibernation were compared with the animals sampled during summer. Muscle tissue were hybridized on a custom 12,800 cDNA probe nylon membrane microarray platform . Six hibernating and six summer active bears were studied in the experiment.