Peripheral circadian gene activity is altered during hibernation in the thirteen-lined ground squirrel.

Many small mammals living in seasonally cold environments rely on hibernation, utilizing strong metabolic rate suppression and a slow consumption of adipose reserves to survive the winter months. The circannual rhythm of hibernation is well known but less is known about the role of the circadian clock while animals are in torpor for weeks at a time. We hypothesized that due to strong global suppression of transcription and translation in the torpid state, that circadian clock activity would likewise be suppressed in peripheral tissues during hibernation. However, the present study indicates that peripheral circadian clock activity persists during torpor. Using 13-lined ground squirrels (Ictidomys tridecemlineatus) as the model, this study analyzed transcript and protein responses by clock components, comparing euthermic control animals with squirrels in deep torpor for >3 days (subcutaneous body temperature 5-8 °C). The data show tissue specific responses by mRNA transcript levels: (a) no significant changes in transcript abundance in liver of control versus torpid squirrels, (b) a strong increase in Nr1d1 levels in white adipose during torpor, and (c) five significant transcript changes in skeletal muscle during torpor (increased Bmal1, Clock, Cry1 and Nr1d1 but decreased Per1). Levels of core clock proteins (BMAL1, CRY2, PER2, and casein kinases CK1δ and CK1ε) were also assessed across five time points of the torpor/arousal cycle showing both tissue- and time-dependent changes in clock proteins that were most prominent in liver and white adipose and indicating that peripheral clocks are still active in tissues over the torpor/arousal cycle.

The Ratio of Linoleic and Linolenic Acid in the Pre-hibernation Diet Influences NFκB Signaling in Garden Dormice During Torpor.

The fatty acid composition of a pre-hibernation diet can influence the depth and duration of metabolic suppression achieved by hibernators. More specifically, a diet high in n-6 polyunsaturated fatty acids (PUFAs) relative to n-3 PUFAs is essential to maximize torpor expression. However, few studies have investigated how diets with different n-6/n-3 PUFA ratios change stress-inducible cell signaling. Garden dormice (Eliomys quercinus) were fed one of three diets designed with different ratios of n-6 PUFA linoleic acid (LA) and n-3 PUFA linolenic acid (ALA). Then, NFκB signaling was assessed in the white adipose, brown adipose, and liver tissues of euthermic and hibernating dormice via multiplex and RT-qPCR analyses of relative protein and transcript levels, respectively. Dormice fed a high LA diet regulated NFκB signaling in a protective manner in all tissues. NFκB signaling was generally decreased in the high LA group, with significant decreases in the protein levels of NFκB mediators IKKα/ß, IκBα, and downstream pro-apoptotic protein FADD. Liver and white adipose from torpid dormice fed a high LA diet increased sod2 expression relative to the other diets or relative to euthermic controls, indicating protection against ROS generated from potentially increased ß-oxidation of n-6 PUFAs. The low LA diet increased biomarkers for apoptosis relative to other diets and relative to euthermia, suggesting low LA diets may be detrimental to hibernator health. Overall, this study suggests that changes in the ratio of n-6/ n-3 PUFAs in the diet influences apoptotic and antioxidant responses in white adipose, brown adipose, and liver of hibernating garden dormice.

Regulation of Peroxisome Proliferator-Activated Receptor Pathway During Torpor in the Garden Dormouse, Eliomys quercinus.

Differential levels of n-6 and n-3 essential polyunsaturated fatty acids (PUFAs) are incorporated into the hibernator's diet in the fall season preceding prolonged, multi-days bouts of torpor, known as hibernation. Peroxisome proliferator-activated receptor (PPAR) transcriptional activators bind lipids and regulate genes involved in fatty acid transport, beta-oxidation, ketogenesis, and insulin sensitivity; essential processes for survival during torpor. Thus, the DNA-binding activity of PPARα, PPARδ, PPARγ, as well as the levels of PPARγ coactivator 1α (PGC-1α) and L-fatty acid binding protein (L-FABP) were investigated in the hibernating garden dormouse (Eliomys quercinus). We found that dormice were hibernating in a similar way regardless of the n-6/n-3 PUFA diets fed to the animals during the fattening phase prior to hibernation. Further, metabolic rates and body mass loss during hibernation did not differ between dietary groups, despite marked differences in fatty acid profiles observed in white adipose tissue prior and at mid-hibernation. Overall, maintenance of PPAR DNA-binding activity was observed during torpor, and across three n-6/n-3 ratios, suggesting alternate mechanisms for the prioritization of lipid catabolism during torpor. Additionally, while no change was seen in L-FABP, significantly altered levels of PGC-1α were observed within the white adipose tissue and likely contributes to enhanced lipid metabolism when the diet favors n-6 PUFAs, i.e., high n-6/n-3 ratio, in both the torpid and euthermic state. Altogether, the maintenance of lipid metabolism during torpor makes it likely that consistent activity or levels of the investigated proteins are in aid of this metabolic profile.

Hibernation impacts lysine methylation dynamics in the 13-lined ground squirrel, Ictidomys tridecemlineatus.

During winter hibernation in mammals, body temperature falls to near-ambient levels, metabolism shifts to favor lipid oxidation, and metabolic rate is strongly suppressed by inhibiting many ATP-expensive processes (e.g., transcription, translation) for animals in order to survive for many months on limited reserves of body fuels. Regulation of such profound changes (i.e., metabolic rate depression) requires rapid and reversible controls provided by protein posttranslational modifications. Protein lysine methylation provides one mechanism by which the functionality, activity, and stability of cellular proteins and enzymes can be modified for the needs of the hibernator. The present study reports the responses of seven lysine methyltransferases (SMYD2, SUV39H1, SET8, SET7/9, G9a, ASH2L, and RBBP5) in skeletal muscle and liver over seven stages of the torpor/arousal cycle in 13-lined ground squirrels (Ictidomys tridecemlineatus). A tissue-specific and stage-specific analysis revealed significant changes in the protein levels of lysine methyltransferases, methylation patterns on histone H3, histone methyltransferase activity, and methylation of the p53 transcription factor. Enzymes typically increased in protein amount in either torpor, arousal, or the transitory periods. Methylation of histone H3 and p53 typically followed the patterns of the methyltransferase enzymes. Overall, these data show that protein lysine methylation is an important regulator of the mammalian hibernation phenotype.

Genes of the undead: hibernation and death display different gene profiles.

A degree of regulation continues into death according to post-mortem transcriptome studies, which have identified 'zombie genes' that come alive hours and days after organismal death. We hypothesized that hibernation, representing the closest natural mammalian phenomenon to death, would display similar gene expression profiles. Exploring zombie genes using qPCR and available transcriptomic resources from multiple torpid tissues in 13-lined ground squirrels showed little in common with gene profiles observed following death. Hibernators repress transcription, surviving only on the transcripts required during profound slowdowns of metabolic rate and of most physiological functions, therefore not requiring zombie gene expression that could be the cell's last resort during stress. This is the first study to explore zombie gene responses to a near-death situation in a living system.