Intracellular antioxidant enzymes are not globally upregulated during hibernation in the major oxidative tissues of the 13-lined ground squirrel Spermophilus tridecemlineatus

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Abstract

Hibernating mammals exhibit oxidative stress resistance in brain, liver and other tissues. In many animals, cellular oxidative stress resistance is associated with enhanced expression of intracellular antioxidant enzymes. Intracellular antioxidant capacity may be upregulated during hibernation to protect against oxidative damage associated with the ischemia-reperfusion that occurs during transitions between torpor and arousal. We tested the hypothesis that the 13-lined ground squirrel (Spermophilus tridecemlineatus), upregulates intracellular antioxidant enzymes in major oxidative tissues during hibernation. The two major intracellular isoforms of superoxide dismutase (MnSOD and CuZnSOD), which catalyze the first step in superoxide detoxification, were quantified in heart, brain and liver tissue using immunodetection and an in-gel activity assay. However, no differences in SOD protein expression or activity were found between active and hibernating squirrels. Measurements of glutathione peroxidase and glutathione reductase, which catalyze hydrogen peroxide removal, were not broadly upregulated during hibernation. The activity of catalase, which catalyzes an alternative hydrogen peroxide detoxification pathway, was higher in heart and brain of torpid squirrels, but lower in liver. Taken together, these data do not support the hypothesis that hibernation is associated with enhanced oxidative stress resistance due to an upregulation of intracellular antioxidant enzymes in the major oxidative tissues.

Introduction

Cellular reactive oxygen species (ROS) metabolism has been the focus of intense interest, due in part to the demonstration that overexpression of antioxidant enzymes confers resistance to oxidative stress, disease (reviewed in Solaini and Harris, 2005, Blomgren and Hagberg, 2006) and, in some instances, increased lifespan (eg. Phillips et al., 2000). Amongst the intracellular antioxidant enzymes, the mitochondrial (MnSOD) and cytosolic (CuZnSOD) superoxide dismutase isoforms are perhaps most strongly linked to oxidative stress and disease resistance and to extended lifespan. For example, MnSOD is upregulated in various tissues of the long-lived type 5 adenylyl cyclase knockout mice (Yan et al., 2007), in long-lived Caenorhabditis elegans mutants (Honda and Honda, 2002), and by the polyphenol resveratrol (Robb et al., 2008a, Robb et al., 2008b), which promotes extended longevity in some animals (see Baur and Sinclair, 2006 for review).

Interestingly, superoxide dismutase activities are also upregulated in a number of species as they enter a metabolically depressed dormant state, such as the hypometabolic C. elegans dauer larva (reviewed in Honda and Honda, 2002), and estivating terrestrial snails (Hermes-Lima and Storey, 1995). Whether enhanced antioxidant capacity is a general strategy practiced by animals entering a dormant state remains to be determined, but recent studies have demonstrated enhanced oxidative stress resistance in brain and liver tissue of hibernators (Lindell et al., 2005, Dave et al., 2006, Christian et al., 2008). Also, one of the major intracellular signaling pathways mediating formation of the stress resistant and long-lived dauer larva in C. elegans is regulated similarly in hibernating 13-lined ground squirrels (Spermophilus tridecemlineatus). Akt, which phosphorylates FOXO and thus inhibits its transcriptional activity, is repressed in brain during hibernation (Cai et al., 2004). This suggests that, during hibernation, FOXO would actively stimulate transcription of its target genes, which include those encoding antioxidant enzymes. Therefore, it is important to establish whether intracellular antioxidant enzymes are indeed upregulated in hibernating mammals and play a role in conferring oxidative stress resistance.

Several authors (Hermes-Lima and Zenteno-Savin, 2002, Carey et al., 2003) have suggested that hibernators and estivators upregulate intracellular antioxidant enzymes to protect against ischemia-reperfusion (I-R) injury during torpor-arousal transitions. Even relatively brief periods of I-R can be fatal to cells in highly oxidative tissues such as heart and brain characterized by rapid ATP turnover (see Solaini and Harris, 2005, Blomgren and Hagberg, 2006 for reviews). Transgenic overexpression of MnSOD, which catalyzes the first step in superoxide radical detoxification in mitochondria, can protect murine heart (Chen et al., 1998) and brain (Murakami et al., 1998, Kim et al., 2002) from I-R injury. In contrast, MnSOD deficiency increases vulnerability of both tissues to oxidant-mediated death (Kim et al., 2002, Asimakis et al., 2002). This protective effect may be mediated by the ability of MnSOD to prevent mitochondrial permeability transition and cell death under a variety of stressful conditions (Silva et al., 2005, Solaini and Harris, 2005, Warner et al., 2004). Therefore, hibernators such as the 13-lined ground squirrel might upregulate intracellular antioxidant enzymes to protect highly oxidative tissues from I-R injury during repeated bouts of torpor and arousal. Other antioxidant enzymes also can be protective in this context. CuZnSOD could effectively dismutate superoxide radicals produced in the cytosol by, for example, xanthine oxidase, which is activated in rodents during I-R (Warner et al., 2004). Similarly, transgenic overexpression of other antioxidant enzymes such as glutathione peroxidase and catalase confer resistance to I-R damage in murine brain tissue and might play a similar role during hibernation (Warner et al., 2004).

Several authors have investigated antioxidant enzyme activities in hibernators (reviewed in Carey et al., 2003). However, these studies have generally not focused on brain and heart tissue, though these are most vulnerable to I-R injury in other mammals, or they have quantified SOD activities directly in crude tissue homogenates using spectrophotometry. Spectrophotometric assays of SOD isoforms are particularly problematic, due to the presence of multiple sources of potential interference (Beyer and Fridovich, 1987, Spitz and Oberley, 1989). To address this problem, assays that employ native gel electrophoresis to separate SOD isoforms from interfering molecules prior to quantification have been developed (Beauchamp and Fridovich, 1971). In addition, quantification by Western blot is useful, as neither SOD isoform is known to be physiologically regulated by phosphorylation or by other post-translational modification, so that there is good agreement between the enzyme level and activity. Also, for measurement of MnSOD, it is critical to account for the fact that this isoform is localized exclusively in mitochondria, so that any change in mitochondrial abundance in cells will affect cellular MnSOD levels/activities without changing the enzyme activity per unit mitochondrion. For this reason, it is necessary to establish mitochondrial abundance, or volume density, to correctly interpret the significance of altered MnSOD protein levels and/or activities in crude tissue homogenates.

Here, we have used both immunodetection and an in-gel activity assay method to measure the levels and activities of MnSOD and CuZnSOD in heart, brain and liver tissues of active and torpid 13-lined ground squirrels. In addition, we have measured the activities of enzymes involved in the second step of superoxide radical detoxification, the conversion of hydrogen peroxide (H2O2) to water. Using these approaches we tested the hypothesis that hibernation is associated with an upregulation of intracellular antioxidant enzymes in the major oxidative tissues.

Section snippets

Materials

Chemicals were purchased from Sigma-Aldrich (Oakville, ON, Canada; including Fluka and Caledon) and Bioshop (Burlington, ON, Canada). BioRad protein dye was obtained from BioRad Laboratories, (Hercules, CA, USA). Prestained broad range protein marker was obtained from BioLabs, (New England, MA, USA). MemCode reversible protein stain kit was purchased from Pierce Biotechnology, (Rockland, IL, USA). Antibodies to human CuZnSOD and human MnSOD were purchased from StressGen Biotechnologies (Assay

Results

The approximately 22 kDa MnSOD and 16 kDa CuZnSOD subunits were both identified in mouse and 13-lined ground squirrel tissues (Fig. 1A). MnSOD and CuZnSOD proteins were immunodetected in heart, brain and liver, with actin serving as a loading control (Fig. 1B shows brain). As has been observed in other rodents (Szymonik-Lesiuk et al., 2003, Meng et al., 2007), SOD levels are particularly high in liver tissue, compared to heart and brain. This is likely related to the role of the liver in

Discussion

We tested the hypothesis that hibernating squirrels adapt to the stress of torpor by upregulating antioxidant enzyme activities in highly oxidative tissues like heart, brain and liver. To address this hypothesis, we assessed antioxidant enzyme levels and activities during the summer period when the animals are active and during torpid states of the hibernation season. Few differences were found in the antioxidant enzymes measured between these two states, suggesting that no adaptive

Acknowledgements

This work was supported by an Early Researcher Award (JAS) and Natural Sciences and Engineering Research Council (NSERC) Discovery Grants awarded to JAS and JFS. MMP is supported by an Ontario Graduate Scholarship (OGS). CWP was supported by an NSERC Undergraduate Student Research Award. The assistance of Alvin Iverson and the staff of the Carman Research Station was invaluable. We would also like to thank Alex Gerson, Jamie Drooker and Jason Brown for help in animal husbandry and tissue

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