Genomics Proteomics Bioinformatics 13 (2015) 111–118

H O S T E D BY

Genomics Proteomics Bioinformatics
www.elsevier.com/locate/gpb www.sciencedirect.com

ORIGINAL RESEARCH

Modulation of Gene Expression in Key Survival Pathways During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus
Kyle K. Biggar 1,3,#,a, Cheng-Wei Wu 1,4,#,b, Shannon N. Tessier 1,5,c, Jing Zhang 1,6,d, Fabien Piﬀeri 2,e, Martine Perret 2,f, Kenneth B. Storey
1 2 3 4 5

1,*,g

6

Institute of Biochemistry & Department of Biology, Carleton University, Ottawa, ON K1S 5B6, Canada UMR 7179 Centre National de la Recherche Scientiﬁque, Muse´um National d’Histoire Naturelle, 91800 Brunoy, France Biochemistry Department, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 5C1, Canada Department of Biology, Genetics Institute, University of Florida, Gainesville, FL 32611, USA Department of Surgery & Center for Engineering in Medicine, Massachusetts General Hospital & Harvard Medical School, Charlestown, MA 02129, USA Chemistry and Chemical Engineering Department, Royal Military College of Canada, Kingston, ON K7K 7B4, Canada

Received 13 February 2015; accepted 20 March 2015 Available online 17 June 2015 Handled by Jun Yu

KEYWORDS Daily torpor; Primate hypometabolism; PPAR gamma coactivator; Ferritin; Chaperone proteins

Abstract A variety of mammals employ torpor as an energy-saving strategy in environments of marginal or severe stress either on a daily basis during their inactive period or on a seasonal basis during prolonged multi-day hibernation. Recently, a few Madagascar lemur species have been identiﬁed as the only primates that exhibit torpor; one of these is the gray mouse lemur (Microcebus murinus). To explore the regulatory mechanisms that underlie daily torpor in a primate, we analyzed the expression of 28 selected genes that represent crucial survival pathways known to be involved in squirrel and bat hibernation. Array-based real-time PCR was used to compare gene expression in control (aroused) versus torpid lemurs in ﬁve tissues including the liver, kidney,

* Corresponding author. E-mail: kenneth_storey@carleton.ca (Storey KB). # Equal contribution. a ORCID: 0000-0002-1204-3329. b ORCID: 0000-0001-6370-429x. c ORCID: 0000-0003-2373-232x. d ORCID: 0000-0002-6076-7321. e ORCID: 0000-0001-9316-1935. f ORCID: 0000-0002-3801-0453. g ORCID: 0000-0002-7363-1853. Peer review under responsibility of Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China.
http://dx.doi.org/10.1016/j.gpb.2015.03.001 1672-0229 ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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skeletal muscle, heart, and brown adipose tissue. Signiﬁcant differences in gene expression during torpor were revealed among genes involved in glycolysis, fatty acid metabolism, antioxidant defense, apoptosis, hypoxia signaling, and protein protection. The results showed upregulation of select genes primarily in liver and brown adipose tissue. For instance, both tissues showed elevated gene expression of peroxisome proliferator activated receptor gamma (ppargc), ferritin (fth1), and protein chaperones during torpor. Overall, the data show that the expression of only a few genes changed during lemur daily torpor, as compared with the broader expression changes reported for hibernation in ground squirrels. These results provide an indication that the alterations in gene expression required for torpor in lemurs are not as extensive as those needed for winter hibernation in squirrel models. However, identiﬁcation of crucial genes with altered expression that support lemur torpor provides key targets to be explored and manipulated toward a goal of translational applications of inducible torpor as a treatment option in human biomedicine.

Introduction
A strong depression of metabolic rate is a hallmark of torpor phenotypes in a wide variety of mammals that employ daily torpor or multi-day hibernation [1]. This survival strategy conserves an animal’s energy expenditure when resources, such as food and water, are scarce or abiotic conditions are highly unfavorable for normal life [1]. A number of genomics-based studies have proﬁled the transcriptional regulation of winter hibernation in rodents and black bears, identifying many genes that are differentially expressed during torpor in hibernators [2–4]. Overall ﬁndings from these studies show coordinated regulation of cellular processes that are involved in metabolic functions and cytoprotection that are crucial to maintaining the torpor phenotype. To date, notable changes in transcription have been linked with genes involved in energy and fuel utilization processes, such as cell growth, glycolysis, lipid metabolism, molecular transport, and cellular stress response (e.g., unfolded protein response, apoptosis, cell cycle arrest, and antioxidant defense) [2,3,5–7]. For many years, the repertoire of animal hibernation models has been primarily rodents with some studies of bats, bears and marsupials [1–7]. However, recently, it has been discovered that a few small lemur species from Madagascar undergo metabolic rate depression and descend into daily torpor or even multi-day hibernation to support survival during the dry season when food is scarce [8,9]. To our best knowledge, the gray mouse lemur (Microcebus murinus) and a few other small Malagasy lemurs represent the only primates with this ability. Indeed, hibernation in lemurs is also novel among mammals, which are the only cases of natural hibernation not accompanied by profound reductions in body temperature [8,9]. Hence, lemurs are the closest species to humans that can undergo naturally-induced hypometabolism. To gain insight into the mechanisms of primate torpor, the present study investigated transcriptional regulation in the gray mouse lemur, by examining the expression of a selected group of genes that have been shown to be regulated in rodent hibernation. Previous studies have shown that although the overall rate of gene transcription is reduced during torpor, expression of some selected genes is upregulated [10]. Examples of these genes include those involved in the regulation of lipid metabolism (fatty acid-binding proteins, peroxisome proliferator-activated receptor gamma coactivator), inhibition of carbohydrate catabolism (pyruvate dehydrogenase kinase), as well as those involved in antioxidant defense (Cu/Zn

superoxide dismutase, aﬂatoxin aldehyde reductase, and heme oxygenase-1) [5,10]. Using real-time PCR, the present study analyzed the expression of 28 genes in ﬁve tissues of M. murinus. These genes were chosen based on their biological roles and their historical signiﬁcance in previous hibernation studies (Table 1) [2,3,5–7], which are involved in the regulation of metabolism, apoptosis, hypoxia resistance, protein chaperones and antioxidant defense. By comparing with previous ﬁndings from hibernating rodents, understanding the regulatory pattern of these genes during torpor can help to determine if the molecular signatures seen in primate torpor are similar to those observed among non-primate mammals. Our study is the ﬁrst to provide molecular evidence of transcriptional regulation in primate torpor.

Results
It has been previously demonstrated that mammalian hibernation typically responds with a relatively conserved set of genes that act to adapt the cell to associated stresses. To compare the torpor response of the lemur to that of other mammalian hibernators that are torpid at characteristically-low Tb, we determined the relative expression of genes previously shown to be torpor-responsive in low Tb hibernators (Table 1), in selected lemur tissues using custom RT-PCR arrays designed for primate species. The relative mRNA expression levels of 28 genes were measured in ﬁve tissues including the liver, kidney, skeletal muscle, heart, and brown adipose tissue (BAT), by comparing control (aroused) and torpid lemurs to determine their transcriptional response during primate torpor. The genes chosen for this study were categorized according to their known cellular roles: metabolism (i.e., fatty acid and glucose metabolism), apoptosis, hypoxia, protein chaperones, and antioxidant defense. In liver tissue, the gene expression of ppargc (2.6 ± 0.43fold), ldha (2.95 ± 0.41-fold), gadd45a (2.11 ± 0.22-fold), and fth1 (2.01 ± 0.39-fold) was found to increase signiﬁcantly during torpor, when compared to controls (Figure 1; P < 0.05). No signiﬁcant changes were observed in any of the other genes studied. Notably, peroxisome proliferatoractivated receptor gamma, coactivator (PPARGC), the protein encoded by ppargc, functions as a key transcription factor that regulates multiple genes within the lipid metabolism. Upregulation of ppargc would thus suggest that controlled regulation of lipid metabolism is likely to be important in torpid

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Table 1
Function Metabolism

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Genes analyzed in this study and their known roles in mammalian hibernation
Gene symbol fabp1 Protein name Fatty acid binding protein 1 Species examined Little brown bat (Myotis lucifugus), Thirteen-lined ground squirrel (Ictidomys Tridecemlineatus), Arctic ground squirrel (Urocitellus parryii) M. lucifugus, I. tridecemlineatus I. tridecemlineatus U. parryii U. parryii U. parryii U. parryii U. parryii Golden hamster (Mesocricetus auratus) I. tridecemlineatus I. tridecemlineatus I. tridecemlineatus M. lucifugus, I. tridecemlineatus U. parryii Richardson’s ground squirrel (U. richardsonii) U. parryii, I. tridecemlineatus I. tridecemlineatus U. parryii U. parryii U. parryii, I. tridecemlineatus U. parryii I. tridecemlineatus U. parryii U. parryii European ground squirrel (I. citellus) I. citellus U. parryii, I. tridecemlineatus U. parryii, I. tridecemlineatus Refs. [11,25–28]

ppargc pdk4 pdk1 ucp1 cpt1a acat1 akr7a2 Apoptosis bax bcl2l1 cdkn1a cdkn1b hif1a slc2a1 hk1 ldha pklr Protein chaperones hspb1 hspa8 hsp90b1 dnajb1 pdia3 gadd45a fth1 sod1 sod2 prdx1 prdx2

Peroxisome proliferator activated receptor gamma, coactivator 1 alpha Pyruvate dehydrogenase kinase, isozyme 4 Pyruvate dehydrogenase kinase, isozyme 1 Uncoupling protein 1 Carnitine palmitoyltransferase 1A Acetyl-CoA acetyltransferase 1 Aﬂatoxin B1 aldehyde reductase member 2 Bcl-2-like protein Bcl-2-like protein Cyclin-dependent Cyclin-dependent 4 1 kinase inhibitor 1A kinase inhibitor 1B

[5,29] [30] [27] [31] [27] [11,27] [11] [32] [33] [34] [34] [15,16] [11] [35] [11,28] [28] [11] [11] [11,28] [11] [28] [11] [11] [36] [36] [11,21] [11,21]

Hypoxia

Hypoxia-inducible factor 1 alpha Glucose transporter protein type 1 Hexokinase 1 Lactate dehydrogenase A Pyruvate kinase, L and R type Heat shock 27 kda protein Heat shock 70 kda protein Heat shock 90 kda protein beta, member 1 Heat shock 40 kda protein Glucose regulated protein, 58 kda Growth arrest and DNA-damage-inducible, alpha Ferritin, heavy polypeptide 1 Superoxide dismutase 1, soluble Superoxide dismutase 2, mitochondrial Peroxiredoxin 1 Peroxiredoxin 2

Antioxidant defense

Note: Urocitellus parryii and U. richardsonii is the synonym of Spermophilus parryii and S. richardsonii, respectively, whereas Ictidomys tridecemlineatus and I. citellus is the synonym of Spermophilus tridecemlineatus and S. citellus, respectively.

regulation of the lemur. In kidney tissue, a signiﬁcant increase in hk1 expression (8.97 ± 1.24-fold) was observed, along with a signiﬁcant decrease in the expression of genes involved in apoptosis and heat shock response including bax (to 49 ± 9% of controls), bcl2l1 (33 ± 3% of controls), gadd45a (50 ± 6% of controls), hspb1 (44 ± 16% of controls), hsp90b1 (29 ± 9% of controls), and pdk4 (10 ± 4% of controls) (Figure 2; P < 0.05). Importantly, hk1 regulates the ratelimiting step in glycolysis. Its upregulation could implicate a potential change in glycolytic ﬂux in the kidney, suggesting possibly differential preference of metabolic fuels between tissues during torpor. In contrast to liver and kidney tissues, no signiﬁcant change in gene expression was found in response to torpor in either the skeletal muscle (Figure 3), or heart (Figure 4) tissues. It could be possible that the relative short duration of lemur torpor (compared to other hibernators) might not be sufﬁcient to induce signiﬁcant changes in expression of genes examined in this study. This would suggest that the torpor-adaptive responses in the muscle tissues are likely unique in lemurs when compared to other hibernators.

In BAT, the expression of dnajb1 (1.87 ± 0.16-fold), fth1 (1.82 ± 0.05-fold), hspb1 (2.61 ± 0.28-fold), and ppargc (1.78 ± 0.21-fold) were found to increase signiﬁcantly in response to torpor, whereas the expression of cdkn1a (42 ± 10% of controls) was found to decrease signiﬁcantly during torpor (Figure 5; P < 0.05). Overall, the transcriptional proﬁling of genes showed tissue-speciﬁc patterns in key cellular processes that regulate metabolic functions and stress responses.

Discussion
This study of gene expression is poised as one of the initial steps in comparing classical models of torpor-hibernation (e.g., squirrels and bats) to the torpor-response of a primate (i.e., the lemur). The genes selected for analysis in the present study were previously shown to be involved in the torpor response of hibernating arctic ground squirrels (Urocitellus parryii), a species that can sustain continuous torpor for many days/weeks with core body temperature falling as low as À3 °C

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Figure 1 Relative transcript expression of 28 selected genes in liver of gray mouse lemurs Histogram shows expression levels of different genes after standardization against b-actin expression. Data are means ± SEM, for 3–4 independent samples from separate animals. * Indicates signiﬁcant difference from the corresponding control (P < 0.05). The genes analyzed are listed in Table 1.

Figure 2 Relative expression of the 28 genes in kidney of gray mouse lemurs Expression levels of different genes were normalized against b-actin expression. Data are means ± SEM, for 3–4 independent samples from separate animals. * Indicates signiﬁcant difference from the corresponding control (P < 0.05).

[11]. By contrast, the gray mouse lemurs analyzed in the present study were held under conditions where they exhibited daily torpor during their non-active hours (they are nocturnal) and showed relative small decreases in body temperature (nadir Tb was 30.8 ± 1.6 °C for the 4 animals in the torpor group). Interestingly, we observed unique patterns of tissuespeciﬁc gene expression in the torpid lemur, when compared to a classical model of mammalian hibernation. Although only expression of some selected genes was upregulated during lemur torpor, many of the proteins encoded by these genes play important survival and adaptational functions.

A previous study documented the expression of heat shock proteins (HSPs) during torpor in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) [12]. A signiﬁcant change in the protein expression of HSP70 was detected in the liver, kidney, heart, intestine, or skeletal muscle between active and torpid squirrels. In addition, the abundance of 75 kDa glucose regulatory protein (GRP75) protein was found to be signiﬁcantly higher in liver, skeletal muscle, and intestine of torpid squirrels when compared to active squirrels [12]. Nonetheless, no signiﬁcant changes in hsp70, also known as (AKA) hspa8, mRNA expression were detected between

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Figure 3 Relative expression of the 28 genes in skeletal muscle of gray mouse lemurs Expression levels of different genes were normalized against b-actin expression. Data are means ± SEM, for 3–4 independent samples from separate animals.

Figure 4 Relative expression of the 28 genes in the heart of gray mouse lemurs Expression levels of different genes were normalized against b-actin expression. Data are means ± SEM, for 3–4 independent samples from separate animals.

euthermic and torpid states in any of the examined tissues of the gray mouse lemur (Figures 1–5), which is different from the signiﬁcant HSP70 protein response seen in torpid thirteen-lined ground squirrels. It should be noted that the torpor-responsive differences in expression of this chaperone between the two species may result from a decrease in HSP70 auto-proteolysis during periods of cold torpor in the hibernator, and not from changes in gene expression [13]. Interestingly, expression of two other chaperone-encoding genes, including dnajb1 (AKA hsp40) encoding the partner protein of HSP70 and hspb1 (AKA hsp27), was elevated in response to torpor in BAT, the thermogenic tissue

(Figure 5). This may be a reﬂection of the necessary physiological role that BAT plays in metabolism and energy supply, along with regulating non-shivering thermogenesis during arousal from torpor. The hypoxia inducible factor (HIF-1) transcription factor is well known to respond to low oxygen levels and has been established to function in the adaptation and protection of cells to hypoxia-related damage [14]. The protection role of HIF-1 includes the upregulation of genes that are necessary to improve oxygen delivery and to enhance the cellular rate of anaerobic glycolysis [15]. A recent study reported differential hif-1a gene expression in the little brown bat (Myotis

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Figure 5 Relative expression of the 28 genes in BAT of gray mouse lemurs Expression levels of different genes were normalized against b-actin expression. Data are means ± SEM, for 3–4 independent samples from separate animals. * Indicates signiﬁcant difference from the corresponding control (P < 0.05).

lucifugus) and the thirteen-lined ground squirrel in response to hibernation [16]. These opposing patterns of hif-1a expression suggest dependence on hibernacula and its oxygen availability (i.e., overwintering in caves vs. underground burrows), rather than a torpor-speciﬁc response [16]. hif-1a mRNA levels were increased in the skeletal muscle and liver tissues of little brown bat, as well as in the liver tissue of the squirrel. In contrast with these hibernators, hif-1a mRNA levels did not change during torpor in any of the lemur tissues examined. However, expression of ldha (encoding lactate dehydrogenase), a downstream gene target of HIF-1a, was upregulated in response to torpor in lemur liver (Figure 1), suggesting that the activity of HIF-1a may be regulated post-transcriptionally, such as that reported for other hibernating animals [16], although it is also possible that ldha may be regulated by another transcription factor, such as the cAMP-responsive element-binding protein CREB. Similar to ldha expression, liver was the only tissue to display a torpor-response by gadd45a, which encodes GADD45a. GADD45a is an integral component of the stress response and is typically involved in arresting the cell cycle under stressful conditions, which are not favorable for growth or cause DNA damage [17]. Although no comparable studies exist for other models of torpor, a similar response has been seen to oxygen deprivation in anoxia-tolerant red-eared slider turtles (Trachemys scripta elegans) [18]. In this regard, it is possible that the increased expression of gadd45a is part of the hypometabolic program that is implemented during periods of environmental stress. The generation of free radicals, such as superoxide (OÀ), 2 can be coupled with changes in metabolic rate. Key enzymatic players in the defense mechanism against reactive oxygen species include peroxiredoxins (antioxidant enzymes that play a major role in the decomposition of H2O2 to H2O and O2) and superoxide dismutases (both Mn-SOD, the mitochondrial isoform and CuZn-SOD, the cytosolic isoform) [19,20]. There are also several auxiliary proteins that are involved in the

antioxidant defense system, including ferritin (composed of heavy and light chains), which is commonly induced under stress conditions. Ferritin is an iron storage protein that sequesters iron in cells so as to minimize the free Fe3+ available to catalyze the Haber–Weiss reaction. A recent study explored the regulation of peroxiredoxins and their contribution to antioxidant defense during torpor in thirteen-lined ground squirrels. It was found that the expression of peroxiredoxin (prdx2) mRNA increased by 1.7- and 3.7-fold in the BAT and the heart, respectively, during squirrel torpor [21]. Although our study failed to see any torpor-responsive changes in prdx1 or prdx2 expression in the lemur, a signiﬁcant elevation of fth1 transcripts was identiﬁed in both liver and BAT (Figures 1 and 5). The increased fth1 expression suggests a key role for ferritin in the torpid lemur for iron storage as one mechanism for protection from iron catalyzed oxidative damage. The switch from carbohydrate to lipid based metabolism is one of the most signiﬁcant changes typically observed during torpor [11,22]. In many hibernating species, animal do not ingest food for several months, switch to a primary reliance of stored lipids for fuel, and strictly conserve existing carbohydrate reserves for tissues that cannot utilize lipids. Recent microarray analyses of Arctic ground squirrels have shown increased expression of genes involved in fatty acid metabolism during torpor [11]. In contrast, results from our study found changes in genes associated with an increase in glycolytic rate during torpor in the gray mouse lemur. The gene expression of ppargc, which encodes peroxisome proliferator-activated receptor gamma coactivator, was elevated signiﬁcantly in both liver and BAT of torpid lemurs (Figures 1 and 5). ppargc is associated with PPAR activation, resulting in lipid uptake in adipocytes and non-oxidative glucose metabolism. The increase in ppargc expression in these tissues may function to increase lipid catabolism in BAT, while shutting down pyruvate oxidation in the liver [5]. Additionally, expression of genes such as hexokinase (hk1) and ldha was upregulated in kidney and liver, respectively (Figures 1 and 2). It is possible that an

Biggar KK et al / Differential Gene Expression in Primate Torpor increase in rate-limited enzyme expression is a preparatory change take place for torpor exit when glycolytic rate can increase. Indeed, glycolytic rate is known to increase quickly during arousal in hibernators such as the deer mouse (Peromyscus maniculatus), where the respiratory quotient (RQ) values rise from $0.75 during torpor to $1.0 upon the transition to arousal [23]. As these are direct measurements of metabolic rate and fuel utilization, it is possible that gene changes take place during torpor to accommodate the glycolytic demand during arousal.

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RNA quality was assessed by the 260/280 nm ratio as well as gel electrophoresis on a 1% agarose gel stained with 1· SYBR Green I (Invitrogen) to check for integrity of the 18S and 28S rRNA bands. All RNA samples were diluted to the concentration at 1 lg/ll using DEPC-treated ddH2O. Gene expression analysis A RT2 First Strand kit (Cat. No 330401) and a custom-designed RT2 Proﬁler PCR array (Cat. No 330131) were obtained from SABiosciences (Mississauga, ON). Gene info used for primer design is listed in Table S1. For ﬁrst-strand cDNA synthesis, 1 lg of total RNA was reversely transcribed in a ﬁnal volume of 10 ll according to manufacturer’s instructions. The cDNA was diluted to 100 ll by adding RNase-free water and stored at À20 °C. PCR was performed using a BioRad MyIQ2 thermocycler (BioRad, Hercules, CA). For one 48-well experiment, 1225 ll of 1 · PCR Master Mix and 49 ll of diluted cDNA were combined in a microcentrifuge tube and an aliquot of 25 ll was added to each well. Proﬁler PCR arrays were run as indicated by the manufacturer (SABiosciences). Melting curves were acquired for all samples for quality control purposes. Statistics Endogenous control b-actin (actb) was present on each PCR array and used for data standardization. Each cycle threshold (Ct) was normalized to the Ct of the endogenous control. The comparative DDCt method was used to calculate relative quantiﬁcation of gene expression. Statistical signiﬁcance of the gene expression difference between the control and torpor samples was assessed using the Student’s t-test with SigmaPlot V.11 software. Signiﬁcant differences were determined at P < 0.05.

Conclusion
In summary, the present study provides insight into the regulation of selected genes that are involved the torpor response in species that are studied as models of mammalian hibernation. Overall results for lemurs indicate an increase in the expression of genes that are involved in fatty acid metabolism and antioxidant defense pathways, with limited correlation to the gene expression changes known to accompany hibernation in ground squirrels. These results provide an indication that the molecular mechanisms of torpor may not be as conserved as previously assumed. Future studies utilizing transcriptomic analysis of gene expression in the gray mouse lemur could greatly improve current scientiﬁc knowledge in the ﬁeld and identify key targets for manipulation if research on natural models of hypometabolism is to be translated into applications that could improve human health.

Materials and methods
Animals Gray mouse lemurs used for this study consisted of 8 adult females (2–3 years of age) born in the authorized breeding colony at the National Museum of Natural History (Brunoy, France; European Institution Agreement # D91-114-1). All experiments were carried out as described in greater detail by Biggar and his colleagues [24]. Brieﬂy, control (aroused) animals were sacriﬁced at the end of a daily torpor bout (i.e., after spontaneous rewarming, Tb 35–36 °C), whereas torpid mouse lemurs were sacriﬁced during a torpor bout (when Tb was at its minimum, 30–33 °C). All animal experiments were performed in accordance with the Principles of Laboratory Animal Care (National Institutes of Health publication 86–23, revised 1985) and the European Communities Council Directive (86/609/EEC). Tissue samples were rapidly excised and immediately frozen in liquid nitrogen. Frozen samples were packed in dry ice and air freighted to Carleton University where tissues were stored continuously at À80 °C until use. All imported tissues were logged as per the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) regulations (export permit No: FR1009118231-E and import permit No: 10cA02291/QWH). Total RNA isolation Total RNA was isolated from the liver, kidney, skeletal muscle, heart, and BAT of euthermic and torpid lemurs with standard procedures using Trizolä (Invitrogen, Waltham, MA).

Authors’ contributions
All authors contributed to the conception and design of the project and to the editing of the manuscript. MP and FP carried out the animal experiments and KKB, CWW, SNT and JZ conducted biochemical assays. Data analysis and assembly of the draft manuscript was carried out by KBS, KKB and CWW. All authors read and approved the ﬁnal manuscript.

Competing interests
The authors have declared no competing interests.

Acknowledgments
We thank Janet M Storey for editorial review of the manuscript and Laurine Haro and Philippe Guesnet for technical and material assistance in the preparation of the lemur tissue samples. This work was supported by a Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (Grant No. 6793) and a grant from the Heart and Stroke Foundation of Canada (Grant No. G-140005874) to KBS. KBS holds the Canada Research Chair in Molecular Physiology; SNT, KKB, and CWW all held NSERC postgraduate scholarships.

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[19] Hermes-Lima M, Storey JM, Storey KB. Antioxidant defenses and animal adaptation to oxygen availability during environmental stress. In: Storey KB, Storey JM, editors. Cell and molecular responses to stress, vol. 2. Amsterdam: Elsevier Press; 2001, pp. 263–87. [20] Hermes-Lima M, Zenteno-Savin T. Animal response to drastic changes in oxygen availability and physiological oxidative stress. Comp Biochem Physiol C 2002;133:537–56. [21] Morin P, Storey KB. Antioxidant defense in hibernation: cloning and expression of peroxiredoxins from hibernating ground squirrels, Spermophilus tridecemlineatus. Arch Biochem Biophys 2007;416:59–65. [22] Giroud S, Perret M, Stein P, Goudable J, Aujard F, Gilbert C, et al. The grey mouse lemur uses season-dependent fat or protein sparing strategies to face chronic food restriction. PLoS One 2010;5:e8823. [23] Heldmaier G, Klingenspor M, Werneyer M, Lampi BJ, Brooks SPJ, Storey KB. Metabolic adjustments during daily torpor in the Djungarian hamster. Am Physiol Soc 1999;276:E896–906. [24] Biggar KK, Wu CW, Tessier SN, Zhang J, Pifferi F, Perret M, Storey KB. Primate torpor: regulation of stress-activated protein kinases during daily torpor in the gray mouse lemur, Microcebus murinus. Genomics Proteomics Bioinformatics 2015;13:81–90. [25] Eddy S, Storey KB. Up-regulation of fatty acid-binding proteins during hibernation in the little brown bat, Myotis lucifugus. Biochim Biophys Acta 2004;1676:63–70. [26] Hittel D, Storey KB. Differential expression of adipose- and heart-type fatty acid binding proteins in hibernating ground squirrels. Biochim Biophys Acta 2001;1522:238–43. [27] Williams CT, Goropashnaya AV, Buck CL, Fedorov VB, Kohl F, Lee TN, et al. Hibernating above the permafrost: effects of ambient temperature and season on expression of metabolic genes in liver and brown adipose tissue of arctic ground squirrels. J Exp Biol 2011;214:1300–6. [28] Epperson LE, Rose JC, Carey HV, Martin SL. Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation. Am J Physiol Regul Integr Comp Physiol 2010;298:R329–40. [29] Eddy SF, Storey KB. Differential expression of Akt, PPARgamma, and PGC-1 during hibernation in bats. Biochem Cell Biol 2003;81:269–74. [30] Buck MJ, Squire TL, Andrews MT. Coordinate expression of the PDK4 gene: a means of regulating fuel selection in a hibernating mammal. Physiol Genomics 2002;8:5–13. [31] Barger JL, Barnes BM, Boyer BB. Regulation of UCP1 and UCP3 in arctic ground squirrels and relation with mitochondrial protein leak. J Appl Physiol 1985;101:339–47. [32] Mele M, Alo R, Avolio E, Canonaco M. Bcl-2/Bax expression levels tend to inﬂuence AMPAergic trafﬁcking mechanisms during hibernation in Mesocricetus auratus. J Mol Neurosci 2015;55:374–84. [33] Rouble AN, Heﬂer J, Mamady H, Storey KB, Tessier SN. Antiapoptotic signaling as a cytoprotective mechanism in mammalian hibernation. PeerJ 2013;1:e29. [34] Wu CW, Storey KB. Pattern of cellular quiescence over the hibernation cycle in liver of thirteen-lined ground squirrels. Cell Cycle 2012;11:1714–26. [35] Abnous K, Storey KB. Skeletal muscle hexokinase: regulation in mammalian hibernation. Mol Cell Biochem 2008;319:41–50. [36] Vucetic M, Stanic A, Otasevic V, Jankovic A, Korac A, Markelic M, et al. The impact of cold acclimation and hibernation on antioxidant defenses in the ground squirrel (Spermophilus citellus): an update. Free Radic Biol Med 2013;65:916–24.

Supplementary material
Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. gpb.2015.03.001.

References
[1] Storey KB. Out cold: biochemical regulation of mammalian hibernation –– a mini-review. Gerontology 2010;56:220–30. [2] Yan J, Burman A, Nichols C, Alila L, Showe LC, Showe MK, et al. Detection of differential gene expression in brown adipose tissue of hibernating arctic ground squirrels with mouse microarrays. Physiol Genomics 2006;25:346–53. [3] Yan J, Barnes BM, Kohl F, Marr TG. Modulation of gene expression in hibernating arctic ground squirrels. Physiol Genomics 2008;32:170–81. [4] Fedorov VB, Goropashnaya AV, Tøien O, Stewart NC, Chang C, Wang H, et al. Modulation of gene expression in heart and liver of hibernating black bears (Ursus americanus). BMC Genomics 2011;12:171. [5] Eddy SF, Morin P, Storey KB. Cloning and expression of PPAR-c and PGC-1alpha from the hibernating ground squirrel, Spermophilus tridecemlineatus. Mol Cell Biochem 2004;269:175–82. [6] Morin P, Ni Z, McMullen DC, Storey KB. Expression of Nrf2 and its downstream gene targets in hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Mol Cell Biochem 2008;312:121–9. [7] Seim I, Fang X, Xiong Z, Lobanov AV, Huang Z, Ma S, et al. Genome analysis reveals insights into physiology and longevity of the Brandt’s bat Myotis brandtii. Nat Commun 2013;4:2212. [8] Kobbe S, Dausmann KH. Hibernation in Malagasy mouse lemurs as a strategy to counter environmental challenge. Naturwissenschaften 2009;96:1221–7. [9] Dausmann KH, Glos J, Ganzhorn JU, Heldmaier G. Hibernation in the tropics: lessons from a primate. J Comp Physiol B 2005;175:147–55. [10] Morin P, Storey KB. Evidence for a reduced transcriptional state during hibernation in ground squirrels. Cryobiology 2006;53:310–8. [11] Xu Y, Shao C, Fedorov VB, Goropashnaya AV, Barnes BM, Yan J. Molecular signatures of mammalian hibernation: comparisons with alternative phenotypes. BMC Genomics 2013;4:567. [12] Carey H, Sills NS, Gorham DA. Stress protein in mammalian hibernation. Amer Zool 1999;39:825–35. [13] Theodorakis NG, Morimoto RI. Post-transcriptional regulation of HSP70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability. Mol Cell Biol 1987;7:4357–68. [14] Semenza GL. Hypoxia-inducible factor 1 (HIF-1) pathway. Sci STKE 2007;2007, cm8. [15] Morin P, Storey KB. Cloning and expression of hypoxia-inducible factor 1alpha from the hibernating ground squirrel, Spermophilus tridecemlineatus. Biochim Biophys Acta 2005;1729:32–40. [16] Maistrovski Y, Biggar KK, Storey KB. HIF-1a regulation in mammalian hibernators: role of non-coding RNA in HIF-1a control during torpor in ground squirrels and bats. J Comp Physiol B 2012;182:849–59. [17] Liebermann DA, Hoffman B. Gadd45 in the response of hematopoietic cells to genotoxic stress. Blood Cells Mol Dis 2007;39:329–35. [18] Zhang J, Biggar KK, Storey KB. Regulation of p53 by reversible post-transcriptional and post-translational mechanisms in liver and skeletal muscle of an anoxia tolerant turtle, Trachemys scripta elegans. Gene 2013;513:147–55.