Paper towel shredding as a novel, affordable, noninvasive method for detecting arousals in hibernating rodents.

Many research groups explore the regulation of hibernation or compare the physiology of heterothermic mammals between the torpid and aroused, euthermic states. Current methods for monitoring torpor (for example, infrared cameras, body temperature or heart-rate telemetry, and motion sensing) are costly, require specialized techniques, and can be invasive. Here we present an alternate method for determining torpor-bout duration that is cost-effective, noninvasive and accurate: paper towel shredding. In the winter, euthermic thirteen-lined ground squirrels will shred paper towels placed in the cage, but torpid animals will not. The presence of a shredded paper towel, indicating an arousal from torpor, is easily evaluated during routine daily monitoring. In 12 animals over 52 days, this simple technique detected 59 arousals with 100% accuracy when compared with the body temperature telemetry of the same animals. Moreover, this novel method avoids some of the drawbacks of other cheap monitoring systems such as the sawdust technique.

Mitochondrial techniques for physiologists.

Mitochondria serve several important roles in maintaining cellular homeostasis, including adenosine triphosphate (ATP) synthesis, apoptotic signalling, and regulation of both reactive oxygen species (ROS) and calcium. Therefore, mitochondrial studies may reveal insights into metabolism at higher levels of physiological organization. The apparent complexity of mitochondrial function may be daunting to researchers new to mitochondrial physiology. This review is aimed, therefore, at such researchers to provide a brief, yet approachable overview of common techniques used to assess mitochondrial function. Here we discuss the use of high-resolution respirometry in mitochondrial experiments and common analytical platforms used for this technique. Next, we compare the use of common mitochondrial preparation techniques, including adherent cells, tissue homogenate, permeabilized fibers and isolated mitochondria. Finally, we outline additional techniques that can be used in tandem with high-resolution respirometry to assess additional aspects of mitochondrial metabolism, including ATP synthesis, calcium uptake, membrane potential and reactive oxygen species emission. We also include limitations to each of these techniques and outline recommendations for experimental design and interpretation. With a general understanding of methodologies commonly used to study mitochondrial physiology, experimenters may begin contributing to our understanding of this organelle, and how it affects other physiological phenotypes.

Electron transport system supercomplexes affect reactive-oxygen species production and respiration in both a hibernator (Ictidomys tridecemlineatus) and a nonhibernator (Rattus norvegicus).

Across many taxa, the complexes of the electron transport system associate with each other within the inner mitochondrial membrane to form supercomplexes (SCs). These SCs are thought to confer some selective advantage, such as increasing cellular respiratory capacity or decreasing the production of damaging reactive oxygen species (ROS). In this study, we investigate the relationship between supercomplex abundance and performance of liver mitochondria isolated from rats that do not hibernate and hibernating ground squirrels in which metabolism fluctuates substantially. We quantified the abundance of SCs (respirasomes (SCs containing CI, CIII, and CIV) or SCs containing CIII and CIV) and examined the relationship with state 3 (OXPHOS) and state 4 (LEAK) respiration rate, as well as net ROS production. We found that, in rats, state 3 and 4 respiration rate correlated negatively with respirasome abundance, but positively with CIII/CIV SC abundance. Despite the greater range of respiration rates in different hibernation stages, these relationships were similar in ground squirrels. This is, to our knowledge, the first report of differential effects of supercomplex types on mitochondrial respiration and ROS production.

Torpid 13-lined ground squirrel liver mitochondria resist anoxia-reoxygenation despite high levels of protein damage.

Hibernation confers resistance to ischemia-reperfusion injury in tissue, but the underlying mechanisms remain unclear. Suppression of mitochondrial respiration during torpor may contribute to this tolerance. To explore this concept, we subjected isolated liver mitochondria from torpid, interbout euthermic (IBE) and summer 13-lined ground squirrels (Ictidomys tridecemlineatus) to 5 min of anoxia, followed by reoxygenation (A/R). We also included rat liver mitochondria as a non-hibernating comparison group. Maximum respiration rates of mitochondria from torpid ground squirrels were not affected by A/R, but in IBE and summer, these rates decreased by 50% following A/R and in rats they decreased by 80%. Comparing net ROS production rates among groups, revealed seasonal differences; mitochondria from IBE and torpor produced 75% less ROS than summer ground squirrels and rats. Measurements of oxidative damage to these mitochondria, both freshly isolated, as well as pre- and post-A/R, demonstrated elevated damage to protein, but not lipids, in all groups. Hibernation likely generates oxidative stress, as freshly isolated mitochondria had greater protein damage in torpor and IBE than in summer and rats. When comparing markers of damage pre- and post-A/R, we found that when RET was active, rat macromolecules were more damaged than when RET is inhibited, but in TLGS markers of damage were similar. This result suggests that suppression of RET during hibernation, both in torpor and IBE, lessens oxidative stress produced during arousal. Taken together our study suggests that ischemia-reperfusion tolerance at the mitochondrial level is associated with metabolically suppressed oxidative phosphorylation during hibernation.

Mitochondrial Metabolism in Hibernation: Regulation and Implications.

Hibernators rapidly and reversibly suppress mitochondrial respiration and whole animal metabolism. Posttranslational modifications likely regulate these mitochondrial changes, which may help conserve energy in winter. These modifications are affected by reactive oxygen species (ROS), so suppressing mitochondrial ROS production may also be important for hibernators, just as it is important for surviving ischemia-reperfusion injury.

Hibernation is super complex: distribution, dynamics, and stability of electron transport system supercomplexes in Ictidomys tridecemlineatus.

Complexes of the electron transport system can associate with each other to form supercomplexes (SCs) within mitochondrial membranes, perhaps increasing respiratory capacity or reducing reactive oxygen species production. In this study, we determined the abundance, composition, and stability of SCs in a mammalian hibernator, in which both whole animal and mitochondrial metabolism change greatly throughout winter. We isolated mitochondria from thirteen-lined ground squirrels (Ictidomys tridecemlineatus) in different hibernation states, as well as from rats (Rattus norvegicus). We extracted mitochondrial proteins using two nonionic detergents of different strengths and quantified SC abundance using two-dimensional gel electrophoresis and immunoblotting. Rat heart and liver had fewer SCs than ground squirrels. Within ground squirrels, SCs are dynamic, changing among hibernation states within a matter of hours. In brown adipose tissue, Complex III composition in different SCs differed between the torpid and interbout euthermic phase of a hibernation bout. In heart and liver, complex III composition changed between winter and summer. We also evaluated the stability of liver SCs using a stronger detergent and found that the stability of SCs differed; torpor SCs were more stable than the SCs of ground squirrels in other states and rats. This study is the first report of SC changes during hibernation and the first to demonstrate their dynamics on a short timescale.

Arousal from Torpor Increases Oxidative Damage in the Hibernating Thirteen-Lined Ground Squirrel (Ictidomys tridecemlineatus).

AbstractDuring hibernation, especially during arousal from torpor to interbout euthermia (IBE), blood flow changes drastically. In nonhibernating mammals, similar changes during ischemia/reperfusion lead to oxidative damage. We hypothesized that suppression of mitochondrial metabolism during hibernation protects against such damage. We compared markers of oxidative damage and total antioxidant capacity in eight tissues among summer, torpid, and IBE thirteen-lined ground squirrels. Overall, summer tissue had less lipid and protein oxidative damage than tissue from the hibernation season, but DNA damage (in four tissues) and total antioxidant capacity (in all eight tissues) were similar among all groups. During torpor, when mitochondrial metabolism is suppressed, lipid damage in heart, brown adipose tissue, and small intestine was lower than IBE by as much as fivefold. By contrast, oxidative damage to protein was at least twofold higher in liver and skeletal muscle in torpor compared with IBE. Our findings suggest that arousal from torpor creates oxidative damage similar to ischemia/reperfusion injury but that this damage is repaired during IBE. These differences cannot be explained by changes in antioxidant capacity, so they are likely due to differences is reactive oxygen species production among hibernation states that may relate to the well-characterized reversible suppression of mitochondrial metabolism during torpor.