Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates

Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates Editors

Anna Nowakowska Michał Caputa N. Copernicus University, Institute of General and Molecular Biology, Department of Animal Physiology, Gagarina Street 9, 87-100 Toruń, Poland

Research Signpost, T.C. 37/661 (2), Fort P.O., Trivandrum-695 023 Kerala, India

Published by Research Signpost 2011; Rights Reserved Research Signpost T.C. 37/661(2), Fort P.O., Trivandrum-695 023, Kerala, India Editors Anna Nowakowska Michał Caputa Managing Editor S.G. Pandalai Publication Manager A. Gayathri Research Signpost and the Editors assume no responsibility for the opinions and statements advanced by contributors ISBN: 978-81-308-0471-2

Foreword Hypometabolism seems to be the most common and the most fascinating strategy of defence in the animal kingdom. Animals use it to survive when they are exposed to extreme conditions such as severe food or water shortages, salinity and pH extremes, cold and a severe shortage or lack of oxygen. As a rule, hypometabolism is temperature-dependent, which means it is, at least in part, a consequence of Q10 effect. Therefore, hypometabolism in ectotherms exposed to cold may be regarded as an opportunistic response. However, in homeotherms it is a regulated process. The regulated decrease in body temperature can be used as a preadaptation to expected adverse conditions. This concerns not only mammalian hibernation and daily torpor (see chapter 5) but also long-term protection against asphyxia in new-born mammals (see chapter 6).

Another form of the regulated decrease in body temperature, universally used in animal kingdom in response to hypoxia, is anapyrexia. Anapyretic level of body temperature is achieved by both autonomic thermolytic responses and coldseeking behaviour (in homeotherms) or by cold-seeking behaviour only (in the majority of poikilotherms). In contrast to the above mentioned opportunistic hypometabolic response of ectotherms to cold, estivating ectotherms are able to use hypometabolism against Van’t Hoff’s-Arrheniuses rule, which means that they manage to reduce their metabolic rate under conditions of increasing ambient temperatures. Moreover, some of them are able to continue estivation for as long as many years.

Animals entering into hypometabolic state down-regulate intracellular energy-demanding processes, such as ion-pumping (chapters 4, 5, and 6) and protein synthesis (chapters 4, 5, 6, 7 and 8). An exception are heat shock proteins (chapters 4 and 8) and enzymes used for antifreeze (chapter 1) and antioxidant (chapters 1, 3 and 4) defence responses. Altogether, hypometabolism seems to be a precisely regulated state exhibiting a lot of features common in the animal kingdom. On the other hand, various groups of animals showing clear-cut differences in physiological mechanisms and living in different environments, present a broad variety of patterns of entrance into and arousal from hypometabolic state. This book focuses on the comparative analysis of the patterns as well as on description of complex genetic, biochemical and physiological mechanisms of adaptation in the main animal groups using hypometabolic strategy of survival. Accordingly, this book deals with peculiar problems encountered by over-wintering and estivating land snails (chapter 1), diapausing insects (chapter 2), marine invertebrates exposed to anoxia, food deprivation, as well as salinity and pH variations (chapter 3), diving turtles,

being unquestionable record-holders among air-breathing vertebrates (chapter 4), mammals entering into hibernation and those using daily torpor (chapter 5), and asphyxiated new-born mammals (chapter 6). The final two chapters thoroughly analyse molecular mechanisms involved in the regulation of hypometabolic state.

Because authors of the chapters are experts in the above mentioned wide variety of topics, the book should be interesting to a broad spectrum of readers.

Michał Caputa

Contents

Chapter 1 Hypometabolism in land snails: Controlled or passive phenomenon?

Anna Nowakowska Chapter 2 Hypometabolism in insects Przemysław Grodzicki and Konrad Walentynowicz Chapter 3 Hypometabolism and antioxidative defense systems in marine invertebrates Eduardo Alves de Almeida and Paolo Di Mascio Chapter 4 Hypometabolism and turtles: Physiological and molecular strategies of anoxic survival Kyle K. Biggar, Amy G. Groom and Kenneth B. Storey Chapter 5 Maintaining metabolic balance in mammalian hibernation and daily torpor James F. Staples Chapter 6 Hypometabolism as a strategy of survival in asphyxiated newborn mammals Justyna Rogalska and Michał Caputa

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Chapter 7 PI3K-Akt regulation as a molecular mechanism of the stress response during aerobic dormancy Jing Zhang, Shannon N. Tessier and Kenneth B. Storey

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Chapter 8 Genetic and epigenetic regulation in hypometabolism Jan Pałyga

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Conclusions and perspectives Michał Caputa

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates, 2011: 1-17 ISBN: 978-81-308-0471-2 Editors: Anna Nowakowska and Michał Caputa

1. Hypometabolism in land snails: Controlled or passive phenomenon?

Anna Nowakowska N. Copernicus University, Institute of General and Molecular Biology, Department of Animal Physiology, Gagarina Street 9, 87-100 Toruń, Poland

Abstract. Hypometabolism in land snails is an adaptive mechanism enabling survival in unfavourable environmental conditions such as the cold, frost, heat and drought. Both estivation and winter torpor are evoked by changes in ambient temperature, and humidity.

Moreover, winter torpor is evoked by a decrease in photoperiod. But there is a growing body of evidence that maintenance in hypometabolic state and arousal from the torpor is under endogenous control, which prevents cell injury induced by extracellular freezing in winter and oxidative stress in both winter and summer.

Hypometabolism in land snails is associated with molecular changes such as gene expression, selective inhibition of some protein synthesis, and changes in activities of same enzymes.

1. Introduction The majority of biological processes are well-ordered in time. They are repeated in specific intervals in diurnal and/or annual sequences. Many Correspondence/Reprint request: Dr. Anna Nowakowska, N. Copernicus University, Institute of General and Molecular Biology, Department of Animal Physiology, Gagarina Street 9, 87-100 Toruń, Poland E-mail: noann@umk.pl

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morphological, biochemical and physiological variables are controlled by endogenous clock mechanisms. The full synchronisation of the organism with environmental conditions promotes survival in changing habitat.

The main component of environmental conditions, which changes daily and/or seasonally, is temperature. Particularly dangerous for life of many endothermic and ectothermic animals is temperature below freezing point.

Therefore, some animals prepare to survive the cold by morphological adaptations, e.g. changes in colour and density of fur but also by physiological adaptations such as changes in metabolic rate. Both the morphological and physiological adjustments are under genetic control.

Nevertheless, environmental changes are necessary to start the genetic expression.

Homeothermy enables birds and mammals to continue active live in cold environment. Body temperature of homeotherms is high and remains relatively independent of ambient temperature. On the other hand, heterothermic vertebrates and invertebrates disappear from ground surface in autumn, when ambient temperature drops. Many species of invertebrates spend the winter under water, where temperatures are typically stable at 0-4째C. Others seek land shelters to hibernate. Some insects stay in exposed sites over winter. They spend the winter under the bark of trees, some bore deep into the wood core of trees, and pupae of various butterflies and moths are hidden in various cracks and crevices. Other insects live inside galls on the stems of woody plants [1].

A particular form of adaptation to survive the cold season is migration. It is characteristic not only of birds but also of invertebrates such as butterflies and land snails. A few butterfly species cover extremely long distances.

Monarch butterflies migrate from all over North America to winter in a small region of the central Mexican mountains. Migration on extremely short distances is observed in land snails. Helix pomatia snails seek appropriate places for burrowing themselves. They dig as much as 0.2 m underground and stay there till the spring [1]. Both the local migration in snails and the long-distance migration in insects are components of the preparation for surviving the winter. They prove that overwintering in invertebrates is not merely a passive phenomenon but a seasonally-phased overwintering strategy.

Impact of the cold on metabolism, coupled with an interruption of feeding, digestion and motor activity, contributes to a general quiescent state over the winter months in the majority of poikilotherms [1]. For many animals the best defence against the unfavourable environmental conditions is ability to reduce their metabolic rate as a results of entering into a hypometabolic dormant state. The ability to survive winter in

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hypometabolic state is a common adaptive strategy used by both endotherms and ectotherms.

Depression of metabolic rate is a common physiological strategy, allowing animals to prolong the duration of their tolerance of suboptimal environmental conditions [2,3]. It means that a reduction in metabolic rate below the normal resting value is the response to any of numerous environmental stresses including temperature, desiccation, anoxia, hypersalinity and food deprivation [4]. Mechanisms which depress metabolic rate have been studies and demonstrated in animals exhibiting different survival strategies, such as hibernating mammals, anoxia tolerant turtles, marine and land molluscs and insects. It would appear that hypometabolim is a simple response to changes in environmental conditions but there is a growing body of evidence that metabolic rate depression is also under endogenous control.

Land invertebrates face unfavourable conditions not only during winter, when temperature drops below 0째C, but also in the temperate zone summer during long periods of dry weather or in the tropical zone during the dry season. Therefore, they spend much time in inactive hypometabolic state not only during winter but also in the summer time. In this chapter I would like to focus on hypometabolism accompanying both winter and summer torpor.

2. Terminology; Classification of hypometabolic states Depending on species and season metabolic adaptation relaying on metabolic rate depression is named hibernation, overwintering, torpor, estivation, diapause or dormancy.

Long hypometabolic state, hibernation, and the short one, daily torpor, are found in at least eleven mammalian orders and in six avian families.

These states, due to lowering whole-body metabolic rate (usually recorded as mass-specific oxygen consumption rate) and allowing body core temperature to fall, are effective strategies for coping with cold environments and limited food availability. Hibernating animals can conserve up to 88% of the energy that would be required to remain active in a typical endothermic manner [5].

Generally, the term hibernation is reserved for true endothermic mammalian hibernators. Hibernation is a highly regulated set of events that provides a considerable conservation of energy stores during periods of an extremely high metabolic demand at a strongly reduced food availability [6].

Preparing for hibernation consists of morphological, hormonal, biochemical and behavioural adjustments. Hibernation appears seasonally and results from changes in photoperiod, temperature, humidity and food availability [7].

These factors influence the start of hibernation but they are not essential, for

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both entering hibernation and the end of this process are precisely regulated by biological clock. During hiberantion body temperature drops almost to the level of ambient temperature but is still precisely regulated. At the end of hibernation the set-point of temperature regulation spontaneously returns to its normothermic level and body temperature rises by non-shivering or shivering thermogenesis [8]. Some of hibernating mammals start the adaptative processes to survive in hypometabolism a long time before entering into hibernation. These are obligatory hibernators. They go into hibernation under stable temperature and photoperiod conditions, which suggests that their torpor is endogenously controlled. Other mammalian hibernators go into torpor when they are food deprived but when they are given food ad libitum they do not hibernate [9]. These are facultative hibernators. In all mammalian hibernators the hibernation season consist of multiple hibernation bouts interrupted by short periods of spontaneous arousal [8].

The term hibernation is sometimes used also to describe winter metabolic depression and torpidity in lower vertebrates. However, these animals belong to ectotherms, which means that they are unable to use midwinter arousals to interrupt their torpidity. Therefore, it is clear that winter torpor in ectotherms is completely different from mammalian hibernation. Literally, the word hibernation refers to inactivity during the winter and it is opposed to estivation, which refers to inactivity during the summer. However, due to the above mentioned controversies, I am going to avoid using the term hibernation to describe winter torpor in invertebrates.

The term estivation, at first glance, seems to be more universal.

However, land snails can stay in estivation for as long as many years [10], which means that the existence of this hypometabolic state is not closely related to the summer season. Estivation is a state of aerobic dormancy that is probably best defined as a survival strategy for dealing with arid conditions.

It is also typically associated with a lack of water and food and frequently with high environmental temperatures [11]. Estivation is commonly used by fish and amphibian, and less commonly by mammalian species, but is the most characteristic of terrestrial snails.

In insects there is a pattern of hypometabolic state which does not fulfil criteria of hibernation or estivation. The state is called diapause [12]. It is a state of arrested development that, depending on the species, can occur at any life stage (egg, larva, pupa, adult) and at any season and that is associated with a reduction of metabolic rate by 70-98%. Diapause usually occurs in response to cold or dry conditions. Because diapause is described separately in one of the chapters of the book I am going to speak about it only marginally.

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3. Mechanisms of hypometabolic responses in land snails Mechanisms of regulation of land snails’ metabolism and survival of the snails in inactive state are still far from being clear. Previous investigations have shown that overwintering in land snails is induced only by external factors such as reduced sources of nutrition, or reduced duration of photoperiod [13] and a drop in ambient temperature [14]. On the other hand, there are some behavioural events used by the snails to prepare to overwinter in cold conditions. To avoid extreme cold exposure they migrate into buffered habitats, withdraw into the shell and some land snails start secreting a thick calcareous epiphragm at the shell aperture. Intertidal snails dig into the mud and freshwater species choose deeper water. There is a growing body of evidence that winter torpor in land snails is precisely controlled by endogenous circannual clock [13, 15, 16]. Blinn [18] regarded hypometabolism as a seasonal strategy enabling winter survival in Allogona snails already in 1963. During their natural overwintering time Allogona snails kept at an elevated ambient temperature were inactive. The author concluded that low ambient temperature is only one of many factors, which induce the dormancy.

However, there is also reason to argue that entering into winter torpor in land snails is a passive phenomenon. Helix pomatia snails given an opportunity to choose ambient temperature in a thermal gradient chamber kept on choosing a warm microenvironment over a period of early to late autumn, long after the last snails had gone into torpor outdoor [16]. This suggests that entering into winter torpor in H.pomatia snails is not facilitated or supported behaviourally.

In molluscs the time of finishing of natural overwintering may be controlled by external factors such as temperature, humidity and light [13] but it may also be controlled by internal factors, for instance accumulated excretory products [13]. The latest studies have shown that there are two more reasons to believe that termination of winter torpor in Helix pomatia snails is a precisely regulated mechanism. Firstly we have shown [16] that winter-torpid snails, maintained in darkness at a constant ambient temperature of 5°C (which is 5°C below the threshold of arousal during the winter), were able to arouse spontaneously in early spring, within a period of natural arousals from torpor in the field. Obviously, they must have been using an internal clock, which does not need any external cues to induce the arousal. Moreover, they must have been able to activate their metabolic reactions under such unfavourable conditions, which is apparently incompatible with their poikilothermic status. Similar ability to arouse from

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torpor at an ambient temperature of 5°C was recorded in the African snail Otala lactea (Muller, 1774) [19]. Secondly, H.pomatia snails showed seasonal changes in concentration of cryoprotectant substances with the highest level of glucose in autumn (before the period of forming the operculum) and the highest glycerol concentration was recorded in winter and spring [20]. The changes were too small to influence freezing point of the snail’s body fluids. Therefore, they might be regarded as rudimentary cryoprotective seasonal responses [20]. One more argument that arousal from winter torpor in land snails is endogenously controlled is their augmented antioxidant defence at the end of the torpor. I address the problem in the last section of the chapter.

Metabolic depression in land snails can be induced not only by cold exposure but also in response to anaerobic conditions or dehydration. During the hot and dry months of summer, snails face a danger of death due to dehydration, and lowered ambient humidity is a cue for starting hypometabolic state in summer [21]. Snails endure arid conditions by retracting into their shells, secrete a thick mucous membrane or epiphragm, over the shell entrance [22], and enter into estivation in which they may survive for months or even years until a return of favourable environmental conditions. During estivation the basal metabolic rate is depressed to a value near 15% of normal [3]. In snails estivation is characterised by their ability to return to active life at a great speed. Stuart et al., [23] have shown that estivating Cepea nemoralis snails maintain activities of most of their enzymes at normal levels that may be an important component of a strategy of the rapid emergence from estivation. Thus, estivation is not a dormancy in the sense that organisms are unresponsive or must undergo major metabolic/ developmental changes in order to return to active life. Altogether, the mechanisms of metabolic suppression in estivators must be (i) rapidly reversible, and (ii) require very little de novo protein synthesis and reorganization of metabolism [11]. Maintenance of elevated antioxidant defence accompanying metabolic depression during estivation is also one of many adjustments preventing oxidative damage during returning to the active state (see the last part of the chapter).

Capacity to survive extended periods in dormant state is a direct consequence of snails’ ability to depress overall metabolic rate to a level that is only 5-30% of that in active snails [24, 25, 26]. Such a profound reduction of metabolic rate in dormant snails seems to be a direct effect of the absence of locomation and an indirect effect of dehydration combined with starvation [24]. The main prerequisites for long-term survival during estivation are water retention and sufficient fuel reserves [11]. In pulmonate land snails

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evaporative water loss during breathing is minimized by apnoic breathing patterns. The snails are able to reduce their breathing rate to only 2-3 breaths per hour and show patterns of discontinuous CO2 release and O2 uptake [25].

An important adaptive response which allows to limit evaporative water loss across the integument is also covering the aperture of the shell with a mucous epiphragm [22].

Although environmental cues for metabolic depression are generally clear, far less is known about molecular signals or mechanisms involved in metabolic depression at a cellular level. Those mechanisms can be divided into two components. One of them shows an intrinsic character, i.e. metabolic depression continues to operate for a long period after tissues are removed from the body. The second concerns extrinsic factors, for example a low extracellular PO2 or high PCO2 , which could induce metabolic depression in animals such as pulmonate snails exposed to a mild hypoxia during estivation. Metabolic depression includes three components of oxygen consumption: nonmitochondrial respiration, mitochondrial respiration driving ATP turnover, and mitochondrial respiration driving proton cycling [27].

Relative contributions to metabolic depression of the extrinsic and intrinsic components should be investigated simultaneously. The putative regulatory mechanisms include: decreased pH, reversible phosphorylation of enzymes to reduce their activity the maintenance of one particular energyutilizing process (ion pumping), and changes in protein synthesis [2, 4, 23].

Some authors suggest that there is the latent mRNA (mRNA pool that is maintained under conditions of large and global inhibition of protein synthesis in a metabolically depressed cell), but its role in the regulation of metabolic depression is not clear [4].

The factor most commonly associated with metabolic depression is cellular acidosis. Entry into estivation is characterised by a sharp and rapid decrease in hemolymph and intracellular pH [28], brought about by periodic apnea and the resultant hypercapnia [25, 29]. Arousal from estivation, which is promoted by a high relative air humidity, is characterised by a return to normal acid-base conditions preceding the rise in metabolic rate [26].

Therefore, a decrease in pH is implicated as an extrinsic factor inducing metabolic depression in land snails [28]. During estivation land snails are withdrawn into their shells and exhibit intermittent breathing episodes. This minimises respiratory water loss. A consequence of the water conservation is a decrease in PO2 and an increase in PCO2, the latter leading to the decrease of pH. The changes in PCO2 and pH are likely to be important signals in the regulation of estivation bout. Indeed, this is not unlike the situation of hibernators. On the other hand, Guppy et al. [2] concluded that a change in intracellular pH was associated with and could be a cause of metabolic

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depression, but proofs of the necessity of a change in pH for metabolic depression or arousal are equivocal.

The role of protein phosphorylation in metabolic depression has been widely examined in estivators. Post-translational modification of enzyme kinetic properties, such as reversible phosporylation and enzyme binding to the cellular particulate fraction, are well established effectors of metabolic regulation in estivating snails [30]. Reversible phosphorylation is the mechanism controlling activities of key regulatory enzymes including pyruvate kinase, pyruvate dehydrogenase and glucose-6-phoshate dehydrogenase (G6PDH). Experiments, performed on Otala lactea snails have shown that entry into estivation results in a strong increase in the phosphorylated fraction of G6PDH. According to a suggestion of Ramnanan & Storey [31] “the properties of phosphorylated G6PDH are consistent with a more active enzyme which may favour enhanced carbon flow through the pentose phosphate cycle during estivation to sustain NADPH production for antioxidant defence�.

It is surprising that during metabolic depression transmembrane Na+/K+ gradients are consistently maintained (depending on type of tissue Na+/K+ ATPases consume 5-40% of a cell energy). However, McMullen and Storey [32] have shown winter suppresion of Na+/K+ ATPases activity in freezetolerant insects Eurosta solidaginis. They have concluded that low temperature reduces the rates of all enzymatic reactions, but the cold can also be a signal for differential regulation of selected enzymes [32]. For example, the cold-induced activation of glycogen phosphorylase that triggers cryoprotectants biosynthesis in cold-tolerant insects arises from differential temperature effects on activities of protein kinase and protein phosphatase that regulate the enzyme so that a net increase in the active, phosphorylated form of the enzyme occurs [33].

Mortality following cold shock is likely to be associated with loss of membrane function as cold shock causes dissipation of transmembrane gradients of Na+ and K+ and a depolarisation of the membrane [34, 35].

The mechanism of hypometabolic defence might also concern an inhibition of protein synthesis. As a matter of fact, protein synthesis is one of the energy-utilizing processes that have been shown to decrease during metabolic depression and whose decrease significantly contributes to the depression [2,3]. Some organisms have been found to decrease the activities of glycolytic enzymes such as pyruvate kinase and phosphofructikinase that are involved in ATP production [36, 37]. On the other hand, the maintenance of most enzymes activities at normal levels may be an important component of the above mentioned strategy, which allows rapid emergence from estivation in land snails [23].

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4. Antifreeze defence during winter torpor At subzero temperatures hypometabolism interferes with survival, due to enhanced risk of frost-bite. Accordingly, land snails living in temperate zone must have developed mechanisms of defence against such a damage.

All ectothermic animals subjected to subzero temperatures are confronted with the vital problem of liquid water in their body. Therefore, they are adapted to the cold by means of a number of morphological, anatomical, biochemical and physiological mechanisms. The biochemical and physiological responses leading to a hypometabolic state have been studied extensively in freeze-tolerant species. Freezing is lethal for most organisms because of ice formation, which destroys subcellular architecture. Invertebrates have developed efficacious cold-hardening mechanisms. The cold hardening is based on a combination of changes in the levels and distribution of ice nucleating agents, accumulation of low molecular solutes such as polyhydric alcohols (polyols) and sugars, and production of antifreeze proteins [38].

There are two main strategies protecting animals against ice formation in their cells: (i) freeze avoidance by enhancing ability of body fluids to supercool and, (ii) freeze tolerance, which means an ability to survive extracellular ice formation in tissues [39]. Both strategies are connected with the following biochemical adaptations: (i) the accumulation of lowmolecular-weight carbohydrate protectants, sugars (trehalose and glucose) and polyols (glycerol, sorbiotol) and (ii) the production of antifreeze proteins (AFPs). The protective low-molecular-weight substances in cold adapted animals can be further divided into two classes based upon their actions: (i) colligative cryoprotectants (which affect vapour pressure or freezing point, depending upon the number of molecules involved) and (ii) cryoprotectants (which stabilize membranes and proteins) [40].

Animals that use freeze avoidance as their winter survival strategy have developed a variety of adaptations that depress both the freezing point (FP) and the supercooling point (SCP, which is temperature of spontaneous crystallization) of body fluids and stabilize the liquid state of their body [1].

Freezing point or melting point is the temperature at which the last tiny ice crystal disappears when a frozen solution is slowely heated. This strategy is particularly common among insects and arthropods (spiders, ticks, mites and isopods) [1]. Freeze-avoiding animals do not tolerate crystalization of their body fluids and thus their supercooling point is equal to their lower lethal temperature. They live, therefore, under constant danger of ice formation in their tissues [39].

Freeze-tolerant animals survive the formation of ice within their tissues.

In these animals ice is allowed to form in extracellular and extraorgan fluid

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spaces, but intracellular water is protected against freezing. They maintain extremely low supercooling point due to the removal of virtually all ice nucleators from the system [41,42]. Freeze tolerance as an adaptation for overwinterig survival occurs widely among terrestrial insects [43-47], nematodes, tardigrades and molluscs [48-52] and also among many vertebrate species [1, 53-55]. In freeze-tolerant species (which survive formation of extracellular ice crystals) sugars and polyols help to alleviate osmotic stress during freezing, as they regulate the amount of water available for freezing and consequently, the extent of cells dehydration caused by extracellular freezing [56]. To control ice formation, freeze-tolerant animals use specific nucleators. Instead of lowering their SCP in winter as freezeavoiding animals do, freeze-tolerant animals raise their SCP by using nucleators, so that freezing begins just below the freezing point [1]. Some animals also appear to have AFPs in their body fluids, which seems contradictory. On the one hand, AFPs inhibit ice formation [1], but on the other it is likely that the function of AFPs in freeze-tolerant animals is to help regulate crystal growth and inhibit recrystalization, the process whereby small crystals regroup over time into larger crystals [1]. Antifreeze proteins do not seem to form aggregates and prevent ice growth by adsorption to the ice surface [38]. Interestingly, genetic analyses have recently revealed that AFPs in some species are derived from selected digestive proteins (such as trypsinogen) that are normally secreted into the gut [1].

The unique property of lowering the freezing point of water (in the presence of ice crystals) without significantly altering the melting point [57] have thermal hysteresis proteins (THPs) which also play an important role in winter survival. Different species of ectothermic animals have different strategies for seasonal regulation of THP production [57]. Some of them start synthesising THPs before the onset of cold weather, others do it only in response to cold weather, whilst still other species synthesise THPs continuously [57]. In the insect D. candensis seasonal expression of THPs is photoperiod dependent and involves secretion of juvenile hormone [57].

Induction of AFPs synthesis in insects is often linked with photoperiod cues and is accelerated by decreasing environmental temperatures [58].

As far as cryoprotectants synthesis in insects is concerned it generally begins in autumn. It is triggered by thermoperiod (extended exposure over several days to temperature at or below a trigger value) but it is facilitated by previous glycogen accumulation in the fat body and an elevation of the activities of the appropriate enzymes, frequently regulated by either developmental or photoperiod cues [58]. The synthesis of polyols and sugars is often triggered only by low ambient temperature [59, 60] but in some insects it is closely related to diapause state [61].

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The insect Eurosta solidagninis uses glycerol and sorbitol as cryoprotectants, which are accumulated according to different seasonal patterns [62]. Initiation of the cryoprotectants synthesis in insects is triggered by the cold so that polyols accumulation can be ended before the insect experiences the subzero temperature [63]. Activities of enzymes associated with cryoprotectants synthesis rise in the fall, whereas activities of enzymes associated with their degradation dominate in the spring, at the time when the cryoprotectants are being catabolised [64, 58]. Moreover, the seasonal acquisition of cold hardiness involves changes in the expression of multiple genes [63]. Altogether, freezing survival relies on a variety of molecular adaptations that include metabolic rate depression, the use of ice nucleators and high concentrations of polyol cryoprotectants, and changes in genes expression [58, 65].

Among insects glycerol is by far the most common cryoprotectant and is produced by both freeze-tolerant and freeze-avoiding species. It induces a decrease of the supercooling point, inhibits the activity of ice-nucleating agents, stabilizes enzymes at low temperatures and prevents water loss [66].

Glycerol and antifreeze sugars are synthesised from a huge reserve of glycogen that is built up during summer and autumn feeding. Enzymes responsible for glycerol synthesis are cold-activated and production of the polyol begins when night time temperatures start to fall below 5째C [1]. The mechanisms which convert glycogen into polyols are regulated by altering the activities of key enzymes of glycolysis in several ways: (i) by seasonal changing the amount of the enzymes, (ii) by direct effect of low temperature on the enzymes kinetics, and (iii) by deactivation through dephosphorylation [67, 68].

The freeze-tolerant frog Hyla versicolor utilizes glucose as a cryoprotectant. The synthesis of this sugar is rapidly initiated upon exposure to temperatures below 0째C at the expense of catabolism of liver glycogen [69]. In contrast, insect species which overwinter in exposed sites and cannot avoid freezing temperatures initiate glycerol synthesis in early autumn well before first frost exposure [70]. This would allow the animal to reduce the major metabolic cost involved in reconverting accumulated glucose to glycogen during the spring [69].

Molluscs are good animal models for studying freeze tolerance. Because land species living in temperate zone have to face huge variations in ambient temperatures (including temperatures markedly below 0째C) they must be able to prepare themselves physiologically for winter before a danger of freezing occurs. According to some authors freeze tolerance in land snails depends on their size [71], emptying of their gut content [72] and decreasing body water content [72, 73]. In land snails, especially in Helicidea, seasonal changes in

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cryoprotectants synthesis and accumulation were recorded, with resulting shifts in their freeze tolerance.

In the land snail Helix aspersa, the supercooling temperature correlated closely with body water content and was negatively correlated with its hemolymph osmolality [52], suggesting that the reduction of total body water content may increase the concentration of the low-molecular-weight compounds and may reduce the amount of freezeable water. It is known that seasonal changes in the temperature of crystalization of body fluids in land snails are associated with shifts in body water content.

Because all animals undergoing freezing face the problem of dehydration of their cells, they need to have physiological and/or biochemical mechanisms that can prevent damage due to dehydration [74]. The reduction of ice formation in the organism limits the dehydration and is of significance in maintaining a critical minimum cell volume [39]. On the other hand, as discussed by Zachariasesen [75], dehydration during winter in terrestrial invertebrates will increase the solute concentration in their body fluids and thereby decrease supercooling point.

Moreover, dehydration has been shown to stimulate polyols production (e. g. glycerol) thus making it difficult to distinguish between physical and biochemical (stimulating) effects of water loss [74]. Trehalose and proline are efficient stabilizers of membrane integrity and protein structure during cooling and dehydration due to ice formation. The mechanisms of stabilization of lipid membranes by trehalose and other sugars seems to rely on preventing the formation of the gel phase in the membranes [39].

Freezing tolerance is an important winter survival strategy for a variety of invertebrates but extracellular freezing combined with low oxygen permeability, through ice covering their body, leads to hypoxic/anoxic state and needs switching to anaerobic metabolism [40]. On the one hand, freezetolerant animals have enhanced their ability to cope with oxygen deprivation, for there is no breathing and no blood circulation while frozen, but on the other, thawing is a reperfusion event associated with the reintroduction of oxygen to tissue, which is likely to induce oxidative damage. It means that thawing leads to transient increase in the levels of reactive oxygen species which imposes oxidative stress.

5. Hypometabolic responses and oxidative stress Land snails are exposed to oxidative stress not only as a result of thawing but also due to arousal from winter and summer torpor. The oxygen radicals are continuously generated by a variety of cellular processes but oxidative stress occurs in living organisms when the rate of generation of oxygen radicals exceeds the rate of their decomposition and such an imbalance leads

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to oxidative damage to biomolecules including lipids, proteins, and nucleic acids. In all animals that spend a lot of time in inactive state, characterised by metabolic suppression, oxidative stress arises not only from low oxygen tension but can also occur during tissues reoxygenation.

Pulmonate land snails can stay immobile and survive in anaerobic conditions not only during overwintering when they are frozen or when they winter underground with their shells closed by operculum but also during both rainy and dry periods in summer. Within a few days of estivation metabolic rate drops to 5-40% of the normal rate [4, 27] and simultaneous inhibition of ventilation imposes mild hypoxic conditions on internal organs [76-78]. Because in land snails oxygen consumption during arousal is six times higher than that in estivating animals [24] this increase in oxygen consumption augments mitochondrial production of ROS [79, 80]. Metabolic rate depression during estivation provides conserving energy on the one hand, but on the other, it imposes hypoxic conditions on internal organs [76-78] and it requires a well-developed antioxidant defence system, enabling the animals to reduce free-radical damage to their tissues during return to the active state [79]. Therefore, one of the most important adaptative mechanisms in land snails is ability to regulate activity of their antioxidant defence system that can minimise free-radical damage to their tissues when breathing resumes after thawing [1] and/or during transition from hypoxic to aerobic conditions during arousal. Antioxidant defence system consists of antioxidant enzymes and low-molecular weight compounds. They reduce the free-radical damage to tissues when animals return to the active state [79]. More details about antioxidant defence system you can find in the chapter 4.

Various vertebrates are able to anticipate oxidative stress by enhancing the defence prior to arousal from winter torpor [81]. There are also some reports concerning similar adjustments in invertebrates [82, 17].

Biological activity of land snails is commonly regarded as fully dependent on thermal conditions. However, Caputa et al. [16], were able to show that arousal from winter torpor and returning from winter hypometabolism in Helix pomatia snails is endogenously controlled. The snails kept in constant darkness at an ambient temperature of 5째C were able to throw their operculum off and aroused from torpor within a period of their natural arousal in the fields. This suggests that also antioxidant defence should be regulated during hypometabolic state. Seasonal changes in antioxidant defence in land snails are well documented but there is no evidence of enhanced antioxidant defence during overwintering in freezetolerant species of snails. On the other hand, land snails are able to maintain redox balance necessary to prevent oxidative damage during arousal from winter torpor [17].

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Surprisingly, although oxygen consumption is significantly reduced during torpor, and the generation of oxyradicals in tissues is generally proportional to oxygen consumption, in some species of land snails the activities of a variety of antioxidant enzymes are elevated during estivation [79], contrarily to the above mentioned lack of changes in winter torpid snails. As suggest by Storey [83] there are three main strategies of antioxidant defence used by animals dealing with wide shifts in the production of reactive oxygen species: first, permanent maintaining of antioxidant defence, the second, rapid induction antioxidant defence response to oxidative stress, and the third strategy would be an enhanced tolerance of accumulation of ROS-damage products combined with an ability of rapid disposal of the products. Some authors have shown that land snails prepare to return to the active state by enhanced activity of some antioxidant enzymes during estivation. Augmented antioxidant activity is a mechanism of preparation for the oxidative stress that accompanies returning of the snails Helix aspersa to activity [79]. Therefore, returning from the hypometabolism to activity seems to be precisely regulated. Moreover, Helix pomatia snails enhance defence against oxidative injury at the end of winter torpor prior to their spontaneous arousal [17], which means that they anticipate oxidative stress accompanying arousal.

Conclusions Taking into consideration all the above mentioned problems it seems that hypometabolic state in land snails is not only passive phenomenon but is also associated with various adaptive mechanisms, which allow survival in cold as well as in dry and hot habitats.

Although entering into hypometabolism is imposed upon the animals by the environmental factors, its maintenance and co-ordination of the vital biochemical pathways can be regarded as a precisely controlled mechanism.

The co-ordination enables the snails to cope with unavoidable oxidative stress accompanying their return to normal activity as well as with extensive freezing accompanying their overwintering.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates, 2011: 19-37 ISBN: 978-81-308-0471-2 Editors: Anna Nowakowska and Michał Caputa

2. Hypometabolism in insects Przemysław Grodzicki and Konrad Walentynowicz Department of Animal Physiology, Institute of General and Molecular Biology, N. Copernicus University, 9 Gagarin St., 87-100 Toruń, Poland

Abstract. Hypometabolism in insects strongly depends on extreme environmental and physiological conditions. In those circumstances hypometabolism is depletion in the Resting Metabolic Rate (RMR) of an insect. It could be caused by several external and internal factors: changes in body mass and body temperature, age, gender, feeding status, reproductive status, time of day, season, latitude, altitude, parasitic infestation, ambient temperature, the ability to flight, and the ability to undergo seasonal endothermy, xeric conditions and diapause.

Diapause is an endogenously controlled, genetically programmed developmental response to changing seasons of the year and environmental conditions. It includes several steps of physiological changes leading to seasonal development arrest. Diapause is the most spectacular example of insect hypometabolism.

Introduction There is no question that one of the most fundamental biological attributes of the organism is its metabolic rate. It affects such basic traits as the energy Correspondence/Reprint request: Dr. Przemysław Grodzicki, Department of Animal Physiology, Institute of General and Molecular Biology, N. Copernicus University, 9 Gagarin St., 87-100 Toruń, Poland E-mail: grodzick@umk.pl

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intake required for the organism to survive and reproduce [1]. According to the definition of Hochachka metabolic rate is a summation of processes which require ATP proceeding on the sub-cellular, cellular and organismic level. The metabolic rate reflects the energetic cost of living under specific environmental conditions [2,3]. It is shaped by complex pathways of energy demand and energy supply, intrinsic (neural and endocrine processes), and extrinsic ecological factors (environmental conditions) under which organisms have evolved to cope and reproduce [4,5].

1. Definition of hypometabolism During their ontogeny insects may increase or decrease their metabolic rate in response to changes in environmental conditions or according to their own developmental needs. In contrast to its medical definition concerning the hypometabolism concept as an abnormal decrease in metabolic rate, insects in definite environmental and physiological conditions can drastically decrease their metabolic rate in a physiological way. Dealing with insects’ metabolic rate we should take into account that generally almost all insects at rest are characterized by relatively low metabolic rates [6]. Hence, it is very important to answer basic questions - what does normal resting metabolic rate mean for insects living in fluctuating environmental conditions, what are the possible factors that can drastically reduce the metabolic rate of these insects and what are the possible ways of regulating it.

2. Factors affecting metabolic rate in insects There are many extrinsic and intrinsic factors that intensify or reduce insects' metabolic rate. The most important factors are temperature and body mass. Moreover, metabolic rate depends on age and ontogeny [5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25], gender [3,24], feeding status [7,8,22], reproductive status [3,24], time of day [3], season [1,3,23,26,27,28,29], latitude (climate) [2,3,5,23,29], parasitic infestation [30], diapause (for details see section 2.2.3), and others.

2.1. Factors increasing metabolic rate The Resting Metabolic Rate (RMR) of insects depends on their body temperature and body mass. The temperature inside the insect’s body achieves equal or close values to the ambient temperature (Ta) [31,32]. The metabolic rate of insects increases linearly with ambient temperature and the

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sensitivity of the dependence i.e. Q10 values may range from 1.5 to 3 [21,31].

It depends on temperature range, interindividual and intrapopulational variation, activity, age, ontogeny, geographical position and other factors [10,22,29,31,33,34].

For a long time an animal’s body mass has been regarded as one of the most important factors affecting its metabolic rate [1]. Traditionally the metabolic rate and the body mass have been related using Kleiber’s allometric equation:

P = aM b With P being metabolic rate, M – the body mass of the animal, b – the mass exponent and a – the mass coefficient [35].

However, in many insects this relationship may be disturbed. In Drosophila melanogaster [1] metabolic rate does not correlate significantly with body mass. Moreover, the correlation may be significant for one season but absent in the following season. While there is a distinct relationship between metabolic rate and body mass when comparisons are made amongst organisms whose mass varies by many orders of magnitude, there also is a large amount of variation in this relationship. Insects can show considerable residual variation in metabolic rate that cannot be attributed to differences in their body size [1].

A very important determinant of the metabolic rate is the current form of the activity accomplished by the organism [3,5,6,11,22,32,36,37]. It comes out especially in the case of energetically costly behaviours, such as flight. It causes a distinct elevation of the metabolic rate highly above its resting value [6,22,37]. In moths the metabolic rate during flight increases by more than 100-fold when compared with their RMR [38]. Resting metabolic rate is directly related to the form of activity that an insect performs currently and depends on its mobility. Insects spending more energy on activity have a higher resting metabolic rate. Accordingly, flying insects in general have a higher resting metabolic rate than species that use energetically less demanding types of locomotion such as walking.

Furthermore, insects producing acoustic advertisement signals are characterized by higher, mass-independent resting metabolic rates [6].

Acoustic insects that pay the largest metabolic cost for signalling have higher RMRs than related non-acoustic species. Relatively high resting metabolic rates are displayed also by species that show endothermy and are able to fly [6,39].

The Table 1 summarizes some examples of the metabolic rate of flying and non-flying insects [modified after 6].

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Table 1. The effect of the form of activity accomplished by the animal (flying or nonflying) on the mass independent resting metabolic rate (ml O2 g-0,75 h-1) in different insect taxes [modified after 6].

FLYING TAXON Blowfly (Order: Diptera) Fruitfly (Order: Diptera) Honey bee (Order: Hymenoptera) Dragonfly (Order: Odonata) Mean value

NON-FLYING RMR 0.658 0.405 2.179 0.591 0.96

TAXON Cockroach (Order: Blattodea) Termite (Order: Isoptera) Ant (Order: Hymenoptera) Cricket (Order: Orthoptera) Mean value

RMR 0.261 0.060 0.122 0.308 0.19

A further factor affecting the metabolic rate of flying insects is their flight ability and flight muscle development. Fully winged representatives of Gryllus firmus have higher metabolic rates than short-winged flightless morphs [6]. Flight ability does not necessarily lead to a high RMR. Winged forms of ants do not have higher RMRs than flightless workers. In this case flight occurs only for a restricted period of time [6].

There is a great amount of data pointing out that a metabolic rate, and its thermal susceptibility, may also be affected by the geographic position of the organism. Cold climate populations living at high latitude or altitude show elevated metabolic rates in comparison with warm climate populations tested at similar ambient temperatures. This phenomenon is often called cold temperature compensation or metabolic cold adaptation (MCA). MCA is beneficial to insects by enabling them to complete growth, development and reproduction at relatively low temperatures [2,3]. Ants living in a cold climate are metabolically more active at low ambient temperatures than ants from southern populations, whereas at high temperatures the situation was reversed [29] (see fig.1).

Figure 1. An illustration of the latitude effect (ants living at 50˚N and 65˚N) on thermal susceptibility of Myrmica ants metabolic activity (MA). Notice clear-cut metabolic cold adaptation (MCA) phenomenon [modified after 29].

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2.2. Factors decreasing metabolic rates 2.2.1. Effect of age and ontogeny The influences of an insect’s age and ontogeny on its metabolic rate have been established in detail in insect larval states. An especially great drop in metabolic rate can be noted between the larva and pupa life stages [9,14,20].

In the case of adult insects there is a great deal of data indicating that metabolic rate decreases with age [40,41] (see fig. 2). This is probably because of the general deterioration of the physiological condition associated with ageing [12]. Some other studies stated no change in metabolic rate [16,20], which may even increase with adult age [11,18,24]. The relationship between age and metabolic rate may also be complicated by indirect internal or external factors, for instance by body mass that also changes with age [11,19]. The negative influence of age on metabolic rate may be explained by respiratory damage of the mitochondria and decrease in the efficiency of the mitochondria to carry out oxidative phosphorylation [10,42,43,44]. Old flies in relation to young change their sensitivity to temperature – Q10 effect. They exhibit a lower metabolic rate than young flies at low temperatures and a higher rate at high temperatures [10,45]. Old fruit flies Drosophila sp. have impaired energy metabolism and are less energy efficient than young flies [45]. Changes in metabolic rate occur most probably due to a disturbance in the activity of cytochrome oxidase that results from changes in the inner mitochondrial membrane. Moreover this effect is probably the result of changes in mitochondrial membrane lipids induced by their peroxidation [10,46,47,48].

Figure 2. The effect of age on metabolic rates in Tse-tse flies Glossina pallidipes [modified after 24] and Colorado potato beetles Leptinotarsa decemlineata [modified after 5].

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2.2.2. Effect of xeric conditions A very important factor affecting the metabolic rate of the insect is the water balance. A reduction in metabolic rate as a response to xeric conditions has been observed. This reduction is surely the mechanism of water conservation ensuing through the reduction in respiratory water loss as a consequence of an alteration in respiratory pattern [2,8,49, 50,51,52,53].

2.2.3. Effect of diapause Xeric conditions mentioned above are one of several environmental factors reducing the metabolic rate of an insect’s organism. The physiology of the organism reflects the correlation between the genome and those environmental impacts. Physiological processes are strongly influenced especially by changes in the environment. Most crucial for survival are seasonal changes in the ambient temperature. Within temperate and polar regions of the earth, the majority of insects avoid these exigencies of the winter through dormancy or migration, which are initiated by a physiological response to the day length called photoperiodism [23,54,55,56,57].

Dormancy is any state of suppressed development, which is adaptive, and usually accompanied by metabolic suppression. It contains two different adaptive processes – quiescence and diapause [15].

Quiescence immediate response, which is not centrally regulated, to a decline in any limiting environmental factor below a particular physiological threshold with immediate resumption of the processes if the factor rises above the threshold [15].

Unlike quiescence, which can occur at any time of the life-cycle, diapause is a more profound, endogenously and centrally mediated, genetically programmed pre-emptive developmental response to changing seasons and environmental conditions, which routes the developmental program away from direct morphogenesis into an alternative diapause programme of a succession of physiological events; the start of the diapause usually precedes the advent of adverse conditions and the end of diapause need not coincide with the end of adversity [15,58].

Many insects have evolved diapause – a strongly evolutionarily preserved developmental programme that has existed in metazoans for millions of years [59].

It represents an alternative developmental pathway, initiated by unique patterns of gene expression that enables insects to survive seasonally recurring chronic forms of environmental stress [60].

This stage of developmental arrest is characterized by processes opposite to those of reproductive growth, such as the arrest or slowing of cell division,

Figure 3. Phases of diapause in insects and links to their development and metabolism [modified after 15].

Hypometabolism in insects 25

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reduction in metabolism and enhancement of stress tolerance [23,60,61,62,63,64].

The two most important functions of diapause are the survival of environmental extremes and the synchronization of reproductive activities with annual cycles of favourable environmental conditions [60]. It could also be responsible for mixing temporally separated genotypes, possibly to advantage in ways that would not otherwise occur [56,61].

Most commonly, insects have a facultative diapause because of their life span. In those animals the environmental cues experienced by individuals, or sometimes their parents during their ontogeny, determines whether an individual will enter diapause [23,55,60]. On the other hand, species having an obligate diapause arrest their development at the same developmental stage every generation regardless of prevailing environmental conditions [58].

Examples of programmed developmental arrest are found in every stage of the insect life cycle from embryonic blastoderm formation to adult reproductive dormancy, illustrating the broad diversity of diapause strategies in insects. The process of diapause occurs at different genetically established developmental stages, but usually at only one life history stage in any given organism [64,65].

Diapause is divided into three main phases: (1) pre-diapause; (2) diapause; and (3) post-diapause quiescence. Each phase may comprise some sub-phases, expression of which depends not only on genotype-driven physiological changes but is also influenced by environmental conditions (see fig. 3). The pre-diapause phase contains the induction and preparation phase; the phase of diapause contains the initiation, maintenance and termination sub-phases [15,60].

Diapause is an environmentally-related process. Typically, at temperate latitudes induction, preparation and initiation take place in late summer and autumn; the maintenance phase occurs in the first part of winter; termination and post-diapause quiescence follow during the latter part of winter and early spring [58].

Induction phase of diapause. Virtually all the insects living in the temperate zone rely on winter diapause [60]. Diapause is induced in advance of the advent of environmental adversity. The entry into and progression through diapause are mediated by molecular mechanisms similar to those that guide cellular reproductive growth; these mechanisms include differential gene expression, post-transcriptional events, post-translational protein modifications and protein localization to specific regions within cells and organs [64].

The decision to enter diapause is initiated when diapause-inducing stimuli are perceived during a fixed and specific sensitive period, which is

Hypometabolism in insects

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genetically determined, and it ranges from various periods within the parental generation through different stages of embryonic, larval and pupal development to the adult individual. The developmental arrest is usually initiated later in the life cycle, and can even occur in the subsequent generation. The induction phase will accrue even when the immediate environmental conditions are favourable to development [66] if adequate token stimuli are received.

In temperate and polar zones, i.e. above 30째N latitude, day length is the primary cue programming diapause. This represents a strong, noise-free indicator of the changing seasons, and has remained highly reliable over evolutionary time [58].The signalling nature of these token stimuli is best understood.

Each individual has its own genetically determined response to day length and, depending on that response, either enters diapause or continues development [66].

For each species of insect there is a critical day length (CDL), which will programme the diapause response. This is designated as the photoperiod that induces a 50% incidence of diapause, and changes with latitude [67].

There are some other specific token stimuli such as pheromonal substances [68,69,70,71], allelochemical substances [72,73] or changes in food quality [74,75] and they are specific to certain conditions (tropics) or even certain species.

In theory, even the environmental factors such as temperature or oxygen level might adopt the role of principal token stimulus in those habitats where they seasonally change in a predictable and sufficiently slow manner, and where photoperiodic or other token signals are less distinct or available (some tropical habitats, soil, caves, deeper layers in large water reservoirs, decaying wood) [15].

In the less common case of obligatory diapause, the initiation of developmental arrest needs no external cues because it represents a fixed component of the ontogenetic programme and is expressed regardless of the environmental conditions. Token stimuli are utilized to induce more widespread facultative diapauses, where individuals can switch between two ontogenetic alternatives, i.e. direct development or diapause [15].

Preparation phase of diapause. After the phase of induction may immediately appear the phase of initiation or those two phases may be more or less separated within the same generation (or even between generations) by a preparation phase.

Diapause induction leads to specific alterations in gene transcription, neuroendocrine milieu and metabolic pathways and the individual is destined

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for later entry into a developmental arrest [76,77]. The preparation phase is the time upon which information about diapause induction is stored.

The preparation phase may be also characterized by different behavioural activities or physiological processes such as migration, location of suitable micro-habitats, aggregation, or the building-up of energy reserves before the final moult/transition into the diapause stage [60].

Initiation phase of diapause is the first of a succession of the above mentioned three eco-physiological sub-phases of diapause: initiation, maintenance and termination. The initiation phase is closely related to the preparation phase, i.e. there can be a migration and reserve storage, but the phase of initiation begins when the ontogenetic stage at which direct development ceases is reached.

When the development is arrested some other processes take place during the initiation phase. One which represents the most general feature of that is the regulated decrease in metabolic rate [23,78].

To establish a suppression of the metabolism there must be changes in the state of metabolic enzymes, the function of biological membranes and gene expression [79,80,81,82,83].

The degree of the metabolism down-regulation differs between species.

Those animals that enter the diapause in a non mobile stage of their development have a relatively rapid decrease in metabolic rate during the initiation phase. On the other hand, in insects diapausing in mobile stages the decrease in metabolic rate is low.

High metabolic activity is required to support specific behavioural and physiological activities. Free living larvae and adults may continue accepting food, [23,55] and actively seek suitable microhabitats [84] or even migrate [85,86].

All those physiological and behavioural activities require a relatively high environmental temperature so it can be accomplished only by permissive conditions [87].

Maintenance phase comes after initiation of the diapause. It is a period when an animal sustains a relatively low and constant metabolic rate and remains locked in developmental arrest. After the initiation phase diapause is maintained even when the environmental conditions are still permissive for direct development continuation. Insects maintain a diapause over several weeks or even months and terminate it by endogenous signal and/or specific change in environmental conditions [15].

Basic processes, such as energy-store depletion and somatic ageing, probably contribute to the gradual change in the physiological state during the maintenance phase. Gradual decreases in diapause intensity and/or increasing sensitivity to diapause-terminating conditions are characteristic

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parameters of the phase [88,89,90]. Some token stimuli (e.g. photoperiod) can prevent diapause from terminating [89].

The duration of diapause can be influenced by many factors: accumulated chilling, moisture, food and day length [23]. For many species, however, a general principle is that diapause duration is shorter at higher temperatures. The reasons for this are not fully understood. One hypothesis is that a higher metabolic rate during diapause at elevated temperatures depletes stored nutrient resources more quickly [65]. Low temperatures are a powerful force reducing the energetic costs of diapause and contribute to the retention of nutrient reserves required for post-diapause processes [58,65].

Coincidently with developmental arrest, diapause is characterized by decreased intermediary and respiratory metabolism [23,55,91]. Metabolic depression involves shutting down or vastly decreasing the activity of energetically expensive biochemical and physiological systems [92,93]. The degree of metabolic suppression varies among insects and correlates roughly with performance requirements of the stage of diapause [65].

On one side, there are various immobile stages such as diapausing embryos, cocooned mature larvae, pre-pupae, and pupae which do not consume any food and display deep metabolic suppression, even if hydrated at relatively high temperatures. On the other side, diapausing free-living larvae and adults can move and their metabolic suppression is usually less pronounced. However there are also extreme cases, where the developmental arrest takes place in otherwise active specimens [15].

Although it may not be obvious in many insects, diapause does not entail a complete cessation of development. As evidenced by characteristic temporal patterns of gas exchange, nutrient metabolism, stress resistance, and gene expression, diapause is a dynamic process [15,60,94,95].

Respiratory and intermediary metabolism adjust to environmental conditions and the generation of molecules required for survival during diapause, the cell cycle reversibly arrests, and stress tolerance is enhanced to protect cell attributes required for resumption of growth and development when diapause terminates [64].

Maintenance of diapause requires specific changes from one type of intermediary metabolism to another, metabolic rate depression and the diversion of energy expenditure away from cellular events required for an active lifestyle [96,97]. To achieve that the diapause-specific gene expression patterns are necessary: (i) those encoding heat shock proteins [98,99,100,101,102,103,104]; (ii) those involved in energy metabolism or energy storage [105,106,107,108]; (iii) those affecting hormonal regulation [76,109,110,111,112,113,114,115,116]; (iv) those encoding clock proteins with

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influence on diapause induction [117,118,119,120,121,122,123,124,125], or (v) those with some other functions [126,127,128,129,130,131,132,133].

Thus, many metabolic pathways, such as the anabolic pathways leading to cell growth and proliferation, are down-regulated during diapause [60,96,134,135]. Other pathways involved in basic cellular maintenance remain operational at reduced levels, and, surprisingly, some pathways are up-regulated during diapause. Most obviously up-regulated are stress resistance pathways leading to cryoprotectants and heat shock proteins synthesis; however, non-stress-related metabolic pathways can also differ [60,96,134,135].

Diapause poses several interesting challenges for nutrient storage and utilization. Commonly, diapauses occurring in the temperate zones last for 9–10 months and, in some less common cases, may persist for a year or more.

Most diapausing insects do not feed at all during diapause or, in the case of some larvae and adults, feed very little. This implies that the insect must sequester sufficient reserves in the pre-diapause period to meet its metabolic needs during diapause and still have sufficient reserves remaining at the end of diapause to complete development and resume activity [65]. For example, after a larval diapause, energy reserves must fuel pupation, metamorphosis, construction of adult tissues, as well as post-diapause feeding and (possibly) reproduction [58,96,136].

Fat reserves are clearly the most important pool used by insects to meet their energy demand during diapause but diapausing and direct-developing insects both store metabolic reserves of the same three major groups of macronutrients: lipids (mainly triacylglycerides [137,138,139,140]), carbohydrates (polysaccharide glycogen [55,140]), and amino acids (the storage hexamerin family of insect proteins [134,141,142]).

Most insects do not feed during diapause because of their life stages such as eggs, pupae, or wandering larvae; therefore all metabolic materials used during diapause originate from reserves. Among those species which diapause in life stages capable of feeding, nutrient intake may be absent, minimized, or distinctly different from that in other periods of their life [23,55].

Even among insects that diapause as adults, nutrient reserves are crucial for restoring post-diapause functions, including the rebuilding of tissues atrophied as part of the diapause programme and for providing energy for post-diapause activities, such as dispersal and reproduction [92,93].

In addition to physiological alterations that decrease metabolic rate, careful selection of a diapause site can affect energetics and costs of diapause. Although depressed, the metabolism remains responsive to temperature and diapausing insects experiencing lower temperatures show

Hypometabolism in insects

31

decreased respiration and slower consumption of reserves than insects experiencing warmer conditions; thus low temperature is a powerful force reducing the energetic costs of diapause [143,144,145].

Termination phase of diapause can start spontaneously and a resumption of development in many species does not require any terminating signals – diapause will terminate even when insects are kept under constant laboratory conditions [111,146,147,148,149,150,151,152,153,154,155,156].

Termination of diapause can be strictly defined and separated from the maintenance phase under laboratory conditions, but in the field the distinction may be not so easy because of its complexity, fluctuations and changes of environmental conditions. Several specific stimuli take place during diapause termination in the field [15].

It is very important to end diapause at a specific time, characteristic for all the population of insects because each individual may initiate diapause during quite different periods of the year and a reliable token signal of diapause termination serves as the synchronizing stimulus.

Termination of diapause means a decrease in diapause intensity to the minimum level and then resumption of direct development is enabled. There are several diapause-terminating conditions [15]. For many wintering species chilling is the most common terminating factor [23,95,157]. Less common, especially in winter diapauses, is the photoperiodic stimuli; nevertheless it is used sometimes [23,158,159]. In some extreme cases wetting [160,161] or drying [162] is expected to terminate diapause.

The process of diapause termination eventually leads to either the overt resumption of direct development (if the conditions are permissive) or the covert return of potentiality for direct development (if the conditions are not permissive) [23,55,88,89,95,157].

Summary The insect’s basic metabolic rate is much less definable than the analoguous factor in homeostatic animals such as mammals for example. The rate of the metabolism in insects depends on several factors which collectively define it. Those factors are internal (for example body mass, body temperature or age) and external (for example ambient temperature, season, photoperiod or humidity). In response to extreme impacts animals could react simply by increasing the activity rate and/or seeking a more comfortable behavioural niche in order to compensate its influence or by reducing their metabolism, which leads to hypometabolism. The most spectacular and most complex hypometabolic response is diapause –

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physiological development arrest. All responses to changing factors independently of their complexity evolved to preserve animals, which leads to survival.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates, 2011: 39-55 ISBN: 978-81-308-0471-2 Editors: Anna Nowakowska and Michał Caputa

3. Hypometabolism and antioxidative defense systems in marine invertebrates 1

Eduardo Alves de Almeida1 and Paolo Di Mascio2 Department of Chemistry and Environmental Sciences, IBILCE-UNESP, São José do Rio Preto São Paulo, Brazil; 2Department of Biochemistry, IQ-USP, São Paulo, São Paulo, Brazil

1. Introduction During normal cell respiration, approximately 0.1–0.2% of the oxygen consumed by aerobic cells is converted into reactive oxygen species (ROS), due to the uncompleted reduction of molecular oxygen in the mitochondrial electron transport chain [1]. ROS includes the superoxide anion radical (O2·-), hydrogen peroxide (H2O2), alkyl peroxides, singlet oxygen (1O2) and the hydroxyl radical (·OH). They are also generated as by-products of several oxygenases, or can be the result of cycle-redox reactions promoted by some pollutants. ROS can be involved in several cellular signaling pathways, including the cell cycle, stress responses, and energy metabolism, and are also important components of defense mechanisms of phagocytic cells [2,3].

However, when in excess ROS can be very deleterious to living organisms because of their ability to oxidize key cell components, such as lipids, proteins and nucleic acids, which sometimes lead to cell death.

Correspondence/Reprint request: Dr. Eduardo Alves de Almeida, Departamento de Química e Ciências Ambientais, IBILCE, UNESP, Av. Cristóvão Colombo 2265, CEP 15054-000, São José do Rio Preto, SP Brazil. E-mail: ealmeida@ibilce.unesp.br

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To protect against the harmful effects of ROS, aerobic organisms have developed a series of antioxidant defenses, to intercept and deactivate ROS before they react with cellular macromolecules. These antioxidant defenses are comprised both enzymatic and non-enzymatic mechanisms, and are present in all forms of aerobic organisms [4].

The three major antioxidant enzymes are the superoxide dismutase (SOD), which decomposes O2•- producing H2O2, catalase (CAT) that decomposes H2O2 to molecular oxygen and water, and glutathione peroxidases (GPx) that decompose H2O2 and organic peroxides, involving in these catalytic process the concomitant oxidation of reduced glutathione (GSH) (Figure 1) [4,5]. Glutathione disulfide (GSSG) should be reduced to GSH by the ancillary flavoenzyme glutathione reductase (GR), which uses NADPH as electron donor [6]. This catalytic cycle lowers the NADPH/NADP+ ratio that in turn should be maintained high in order to prevent oxidative damage. NADPH is recycled by the activity of two enzymes of the oxidative pentose phosphate pathway glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase as well as other enzymes from the intermediary metabolism, such as malic enzyme, isocitrate dehydrogenase [7].

Non-enzymatic defenses include the fat-soluble vitamins α-tocopherol and β-carotene, as well as several low molecular weight compounds, such as ascorbic acid, α-tocopherol and GSH (Figure 2).

Cellular Metabolism

O2-.

SOD H2O2 CAT

GSH

GPx

H2O + O2

NADPH

GR GSSG

G6PDH NADP+

Figure 1. General mechanism of antioxidant and auxiliary enzymes in organisms.

Hypometabolism and antioxidative defense systems in marine invertebrates

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Figure 2. Structure of some non-enzymatic antioxidants.

When the rate of ROS production surpasses the rate of its decomposition by antioxidant defenses and repair systems, a situation called oxidative stress can be established, and levels of oxidative damage usually increase [4]. Marine invertebrates are frequently exposed to natural sources that enhance the formation of ROS, including adverse situations that cause the organism to enter into a hypometabolic state. Entry into a hypometabolic state is an important survival strategy for many organisms when they are challenged by environmental stressors, including low oxygen, cold temperatures and a lack of food or water. Much of the resistance and tolerance of marine invertebrates to hipometabolic periods is believed to be due to the effectiveness of animal antioxidant responses activated during this insult, which provide protection against any oxidative stress that could be generated by this situation. This chapter focuses on some important advances in the understanding of the role of antioxidant enzymes in marine invertebrates subjected to hypometabolism.

2. ROS generation during hypometabolism The study of oxidative stress in animals in a hypometabolic state first started with studies on garter snakes Thamnophis sirtalis parietalis either under anoxia (10 hours, 5˚ C) or exposed to freezing temperatures (5 hours, –2.5˚ C), and on brains of anoxia-tolerant turtles [8-10]. It was shown that snakes are capable of increasing both the activity of SOD and the levels of

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glutathione during anoxia, and are also capable of increasing GPx and CAT during freezing. Rice et al. [10] observed an increase in ascorbate levels in turtles, an increase which was associated with tolerance to hypoxia and reoxygenation stress.

Based on these results, the authors of these initial studies hypothesized that animals that are tolerant to hypometabolism can increase antioxidant defenses or even maintain them at typical levels during anoxia/hypoxia insult as a form of preparation for the potential oxidative stress caused by reoxygenation or thawing [11,12].

Since the publication of these two pioneer studies, many other articles have been published on a large variety of vertebrate and invertebrate animals placed under hypometabolism. The majority of these studies have been related to oxygen deprivation. In most of these studies, authors found elevations in antioxidant defenses and/or upregulation of genes encoding antioxidant enzymes in association with a decrease in oxygen availability.

With regard to marine invertebrates, there are few studies focused on antioxidant capacity under hypometabolic insult, and antioxidant modulation has been also found.

The hypothesis of a preparative mechanism for arousal are widely accepted by researchers in the area today, because it has been proven that, in some cases, these increases in antioxidant enzymes can be maintained during the arousal period, and many of the non-enzymatic antioxidants are rapidly consumed as the oxygen concentration increases in tissues.

For example, studies on ground squirrels have shown that plasma ascorbate concentrations increase three- to fivefold during hibernation, but fall rapidly during arousal with the highest rate of ascorbate use correlating with the peak of O2 consumption during thermogenesis and also correlating with a transient peak in plasma urate (a degradation product of ROS attack) [13].

Indeed, there are evidences that ROS generation decreases during anoxia but increases upon reoxygenation in turtles [14,15] and shrimp (Litopenaeus vannamei) [16]. The absence of a large increase in oxidative damage during reoxygenation has been attributed to the increase in antioxidant enzymes.

However, although these authors suggest that ROS can either be effectively counteracted by antioxidant or does not change during anoxia, in some species, oxidative damage has been found to increase during oxygen deprivation, giving rise to the possibility that ROS can also increase in some species experiencing oxygen deprivation.

Today, studies have repeatedly shown that ROS can be continuously produced during anoxia/hypoxia in different animal models, including marine invertebrates, and it has also been concluded that these ROS are

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important modulators of molecular and cellular responses during hypoxia [17-22].

The main evidence of ROS production during hypometabolism in aquatic organisms is the increase in the levels of oxidative damage to macromolecules, which is elicited by increases in lipid peroxidation products, oxidized DNA bases and oxidized proteins. For example, the Atlantic crab Chasmagnathus granulata showed no variation in the antioxidant enzymes SOD and GST after 8 hours of anoxia followed by 20 and 40 min of air recovery, but lipid peroxidation increased significantly during the anoxia period [23]. This finding clearly shows that ROS can be produced during anoxia and that animals can maintain typical levels of antioxidant defenses to survive in this state. However, there were not enough antioxidant defenses to completely counteract ROS. Additionally, Almeida et al. [24] found that the brown mussel Perna perna shows a significant increase in the level of oxidative DNA damage and lipid peroxidation after 24 hours of air exposure, with no changes in any antioxidant enzymes in the gills or the digestive gland; aside from those changes seen in GST activity which increased in the digestive gland after 18 hour of air exposure. After the animals were re-submersed in water, oxidative DNA damage and lipid peroxidation returned to control values and antioxidant defenses remained at the same level; however, GST still increased. One hypothesis for this increase is that it aids in the excretion of metabolic products generated during the hypometabolic period. This finding further indicates that ROS can be generated during hypoxic periods and also that marine invertebrates can counteract the effects of these reactive species by maintaining active antioxidant defenses, even during low oxygen availability.

It has been proposed that the generation of ROS during anoxia is due to a decrease in the cytochrome oxidase Vmax during the electron transport chain of the mitochondria [25]. This decrease could be responsible for an increase in mitochondrial redox state, which, in turn, accelerates ROS generation during hypoxia, triggering the activation of different transcriptional factors involved in numerous cellular hypoxia responses, and increasing oxidative damage [24].

Interestingly, it has also been reported that marine invertebrates possess alternative proteins for electron transport chain during oxygen deprivation, called alternative end oxidases (AOX, figure 3). They branch off the classical respiratory chain after complex I and transfer electrons from reduced ubiquinone (Coenzyme Q) to AOX, which in turn donates its electrons directly to complex IV to reduce O2 to water [26,27]. The proposed function of AOX has been that it maintain respiratory electron flux, substrate

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Eduardo Alves de Almeida & Paolo Di Mascio

Â

Complex I

Complex IV

Complex III

Intermembrane space

cyt c

Q AOX

Matrix

H2O

NADH + H+

½ O2 +

2H+

NAD+

Figure 3. Schematic diagram of the mitochondrial electron transport chain showing the alternative oxidase pathway promoted by AOX in some marine invertebrates (adapted from Abele et al., [28]).

oxidation and cellular redox potential when the classical cytochrome oxidase is inhibited, and also that it maintain redox balance and intracellular PO2 under ADP limiting, metabolically down regulated conditions, or when O2 levels within the cell start to rise because the classical respiratory chain is O2 saturated [28]. This implies that the AOX system minimizes the risk of oxyradical formation and therefore it can be assigned with an antioxidant function [29]. The AOX gene was recently detected in marine ectotherms from the phyla Mollusca, Nematoda and Urochordata, and expression was confirmed in different tissues of the tunicate Ciona intestinalis and the oyster Crassostrea gigas [30].

3. Antioxidant defenses in marine invertebrates submitted to hypoxia/anoxia A compilation of studies found in the literature on antioxidant defenses in marine invertebrates experiencing hypoxia or anoxia is presented in Table 1. It can be noted that there is very little research on marine invertebrates, indicating a substantial need of more studies in order to better understand the role of these responses in other marine organisms, i.e. possessing different habits or habitats.

During oxygen deprivation antioxidant defenses generally increase or are at least maintained at typical normoxic levels, a response which can be associated with a defense strategy to protect against the ROS that is produced during the hypoxic/anoxic state. This response may also be due to a preparative mechanism for arousal, when oxygen is restored. It is also possible that both hypotheses occur. However, decreases in specific antioxidants have been also described for some species.

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Table 1. Examples of antioxidant defense modulation in different marine invertebrate species submitted to hypoxia or anoxia.

These responses have been studied at different cellular level, such as the analysis of the activity of the antioxidant enzyme, quantification of nonenzymatic component concentration in tissues, the evaluation of expression of antioxidant genes and, most recently, through 2D SDS-PAGE followed by protein sequencing to identify proteins differentially expressed in oxygen deprived animals.

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Studies involving the measurement of antioxidant enzyme activities are generally focused on classical SOD, CAT, GPx and GST enzymes, while the evaluation of non-enzymatic antioxidant is most related to the quantification of GSH and GSSG. Using a proteomics approach, however, Jiang et al. [37] were able to identify other non-traditional antioxidants when studying Chinese shrimp response to oxygen deprivation, identifying a down-regulation of fatty acid binding protein like 10 (FABP10), carbonyl reductase 1 (CBR1), and aldehyde reductase 1(ALDR1).

FABP10 has high affinity and the capacity to bind to long-chain fatty acid oxidative products [41]. CBR1 catalyzes detoxification of the lipid peroxidation products under oxidative stress conditions [42,43]. ALDR1 catalyzes the reduction of several lipid peroxidation and glycoxidation products, including methylglyoxal, 3-deoxyglucosone, acrolein and 4-hydroxy-2-nonenal [44]. The down-regulation of these antioxidant proteins in hepatopancreas of shrimp may reflect a reduced ROS production level after hypoxia treatment in this species.

There are several main factors to be considered from the Jiang’s study.

The first factor is that this species presented a general decrease in antioxidant defenses under hypoxia, revealing a different profile in antioxidant responses to oxygen deprivation. Another factor is that there are other antioxidant enzymes that are not being currently evaluated in any other marine invertebrate species, but that seems to be an important part of the antioxidant responses of these organisms to oxygen deprivation. Thus, even the lack of differences in the activities of classical antioxidant enzymes such as SOD, CAT and GPx during oxygen deprivation does not necessarily means that antioxidant defenses were unaltered. It is possible that several other unknown antioxidants are acting during this situation, but this possibility must be investigated further.

Although the data presented in Table 1 shows that different marine invertebrate species present different antioxidant responses to handle oxygen deprivation, there are many aspects of these responses that call for more extensive studies. The responses activated by marine invertebrate species as a result of oxygen deprivation may actually be dependent on several factors, including the time duration that the organism was exposed to hypoxia/anoxia, the inherent effectiveness of the species to handle periodic variations in oxygen availability in their habitats, the efficacy of the selected antioxidant response, the influence of other environmental factors such as temperature, food availability, salinity and water pH, and maybe also the frequency with which an animal is subjected to these stressful environmental conditions.

Animals enduring daily variations in oxygen availability have developed important strategies for antioxidant modulation as a response to these

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stressful states, contributing to the tolerance to anoxia or hypoxia. Thus, the study of antioxidant responses in oxygen deprivation tolerant and not tolerant animals is also important in order to elucidate how some organisms have developed their adaptations to inhabit stressful environments during the course of evolution.

Oliveira et al. [23] observed that the estuarine crab Chasmagnathus granulate submitted to oxygen deprivation for 8 hours presented a decrease in SOD activity but an increase in CAT and GST in the gill. However, there has been no research into possible changes in these enzymes when the animals are subjected to oxygen deprivation for more or less than 8 h. As has been the case in many other species, the results may change after 2, 4 or 24 hours of oxygen deprivation. A better characterization of these responses in different species may reveal some clues as to how a specific animal are more or less tolerant to anoxia or hypoxia when compared to other species, and may also reveal what strategies these organisms have developed to handle different times of oxygen deprivation.

Intertidal mussels are likely forced to respond to periodic variations in oxygen concentration due to tidal cycles that expose them to air. Previous research would suggest that these mussels have developed important responses during the course of evolution to protect themselves against this adverse state, including the maintenance of antioxidant defenses. As shown in Table 1, the brown mussel P. perna displays an increase in SOD activity after 4 hours of air exposure [31]. However, when exposed for a longer period, SOD was similar to control values, and GST became more active [24]. These results demonstrate a clear differential modulation in antioxidant defense during the time of hypoxia.

Along this vein, Weihe et al. [45] recently found that the intertidal Antarctic limpet Nacella concinna had significantly higher SOD and CAT activities than sub-intertidal limpets, in both gills and digestive glands.

Sub-intertidal animals are constantly immersed in seawater and do not suffer from the effects of tidal oscilation; therefore, their antioxidant defenses are lower. Similarly, Almeida et al. [7] also described that intertidal mussels P. perna have increased antioxidant defenses when compared to farmed mussels that were constantly immersed in seawater.

Another curious induced-hypometabolic state in marine invertebrates related to changes in oxygen availability is observed within some symbiotic marine organisms. Some cnidarian species, for example, in symbiosis with photosynthetic protists (zooxanthellae) must withstand daily hyperoxic/anoxic transitions within their host cells. The O2 produced by photosynthetic cells in symbiosis with animal cells in daylight can put the

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host cells in a hyperoxic state. Contrarily, an anoxic state is observed at night, due to respiration of both the photosynthetic symbiont organism and the animal host cells [46]. As a consequence, symbiotic cnidarians have a higher isoform diversity of SOD genes and also a higher SOD activity when compared to non-symbiotic species [46-48]. It was reported that the symbiotic cnidarian Anemonia viridis presented at least five SOD isoforms and high SOD activity, while the non-symbiotic Actinia schmidti presented only three isoforms and lower SOD activity compared to A. viridis [47], results which were consistent with the hyperoxic/anoxic states of symbiotic organism.

4. Antioxidant defenses in marine invertebrates subjected to food deprivation Another common environmental insult that causes marine invertebrates to enter into a hypometabolic state is food scarcity. Studies have shown that the growth rate of marine Antartic organisms is influenced by both food availability and temperature [49]. Many benthic species cease feeding in winter for periods ranging from a few weeks to many months [50], due to food deprivation. In this case, organisms generally present an overall decrease in metabolic rate in order to better maintain cellular energy when faced with a low food supply. Additionally, it has been demonstrated that some of these Antartic species are faced with starvation period when food is scarce.

Antioxidant defenses can also change in response to starvation. Some authors have proposed that most of the deleterious effects of starvation can be attributed to ROS generation during food deprivation [51,52]. As the oxygen consumption is very low during periods of starvation, animals may cope with oxidative stress using mechanisms that are very similar to those used during periods of oxygen deprivation.

Abele et al. [53] have recorded reduced levels of SOD in the digestive gland and CAT in the gills of N. concinna after one month starvation. Pinho et al. [54] observed a decrease in CAT activity in the hepatopancreas of the Chasmagnathus granulatus after one week of starvation, without any measurable change in SOD activity. Again, both increases and decreases, and even cases with no changes, have been found in the antioxidant status of starved animals. Increases in antioxidants can again be related to a protection against ROS, which can be produced either during or after the starvation period. In this respect, Antartic limpets (Nacella concinna) starved from one to four weeks via food deprivation, presented increased SOD, CAT and GST

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activities [55], which may be connected to the preparative response hypothesis.

Another possibility is that antioxidant enzymes increase as a compensatory mechanism resulting from a deficiency in a diet with low molecular weight components such as ascorbic acid, Îą-tocopherol and carotenoids, which are well known antioxidants in marine algae [56]. A deficit of these compounds could become a critical factor for the antioxidant status of starved animals [53].

The importance of antioxidant maintenance during food deprivation in invertebrates can also be observed when considering the activity of the glucose phosphate shunt enzyme G6PDH. During food deprivation, a general suppression of G6PDH activity is expected because carbohydrate catabolism and fat acid biosynthesis decrease. However, it has been shown that the activity of this enzyme can increase during starvation in order to guarantee the NADPH supply that is needed to maintain antioxidant status. Ramnanan and Storey [57] have identified two distinct G6PDH proteins in the hepatopancreas of the land snail Otala lactea, one of which was predominant in active snails and the other of which was more active in starved animals, which also showed greater resistance to urea denaturation and to moderately high temperatures. Additionally, this G6PDH isoform exhibited a lower Km for the substrate glucose-6-phosphate. Similar studies on marine invertebrates do not exist in the literature.

5. Antioxidant defenses in marine invertebrates subjected to other environmental insults The entry into hypometabolism is sometimes difficult to identify in marine invertebrates. Identification processes generally include the measurement of factors such as oxygen and carbohydrate consumption, intermediate metabolism enzymes and ATP/ADP ratios in animals subjected to adverse environmental conditions. But considering that the study of intermediate metabolism in most marine invertebrate species does not exist even in typical environmental conditions, there is an enormous challenge in recognizing species that withstand hypometabolic periods, as well as in identifying the environmental insults that trigger these responses in each marine species.

In general, hypometabolism can result from a general decrease in metabolic activity due to stressful conditions. Although hypometabolism can be more easily associated or identified in animals experiencing oxygen or

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food deprivation, several other environmental insults can also account for metabolic depression generating a hypometabolic state. These other possibilities include variations in environmental parameters such as pH, salinity and temperature, which must be optimal for each organism. In extreme cases, when the environment loses its optimal conditions, it can instigate the beginning of a starvation processes in some species. Thus, some responses activated during variations in salinity, pH and temperature could be also important in the understanding of the mechanisms preceded by metabolic depression and starvation.

Some studies have found that disk abalones subjected to increased temperatures, low salinity and oxygen deprivation presented significant changes in the activity of several antioxidant enzymes [36], suggesting that salinity and temperature stress can instigate an oxidative stress condition similar to that observed during periods of oxygen deprivation. It is probable that the increases in SOD, CAT, GPx and thioredoxin peroxidase observed by these authors during thermal and salinity stress are a response to ROS generated during exposure to these stressors, but this could be also a preparative response for when the animals return to optimal environmental conditions.

With respect to variations in water pH, there is some research that reports important changes in ROS production and antioxidant modulation, and there is also a study describing a hypometabolic state in a marine gastropod (Littorina littorea) at low seawater pH, evidenced by an overall decrease in metabolic rate and oxygen consumption [58]. Other studies have reported that acidic or alkaline-induced oxidative stress may cause DNA damage, and may cooperatively activate expression of CAT, GPx and TRx mRNA in haemocytes and hepatopancreas cells of shrimp (Litopenaeus vannamei), with more injury in high alkalinity environments than in acidic environments [59]. Another study reported that GST activity also increase in this same species in water at low pH [60].

When the mud crab Scylla serrata was exposed to increased salinity, an overall decrease in oxygen consumption was also observed [61], suggesting that salinity variations, as well as pH variations, can also trigger metabolic depression in marine invertebrates. These authors also observed significant variations in antioxidant enzymes, which may be related to modulations in ROS production during the insult, or which may also be due to a preparative response activated by the decrease in oxygen consumption. Significant changes in antioxidant defenses in response to salinity stress was also observed in the shrimp Litopenaeus vannamei [62], as well as in the intertidal copepod Tigriopus japonicus [63].

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Finally, temperature stress has been also associated with changes in antioxidant defenses in marine invertebrates. It is known that increasing temperature can increase the activity of several enzymes, including antioxidant enzymes in most of animals, both vertebrate and invertebrate, with an opposite effect at low temperatures. But for aquatic species such as marine invertebrates, it is important to note that the temperature has a direct effect on enzyme kinetics and metabolism, but also influences oxygen solubility in water, therefore causing changes in oxygen availability.

It has been proposed that changing environmental temperatures to those outside the optimal temperature range in marine ectotherms causes a decrease in aerobic scope of animals upon both warming and cooling, and can also induce a state of functional hypoxia in fully oxygenated water [64]. In line with findings in several marine invertebrates, Lannig et al. [65] reported that Atlantic cods below the optimum temperature range presented an overall reduction of blood circulation and oxygen transport, which may be responsible for the significant lowering of venous PO2. It has also been shown that hypoxia inducible factor 1 (HIF-1), which trigger the transcription of target genes involved in hypoxia responses (for review see [66]), can also be activated during stressful exposure to cold and recovery in the North Sea eelpout (Zoarces viviparus) [67].

Previous studies has indicated that temperature change has a considerable influence on physiological stress responses in aquatic organisms, and that rises in temperature leads to higher metabolism which increases oxygen consumption and consequently increases ROS production [68-70]. In this case, an increase in antioxidant enzymes can commonly be observed.

Conversely, when exposed to moderate or low temperatures, antioxidant defenses can decrease following the decrease in the metabolic rate and, consequently oxygen consumption decreases. However, antioxidant defenses can also increases at low temperatures. For example Zhou et al. [71] observed that the expression of the enzymes SOD, CAT, GPx and GST increased significantly as the temperature increased from 22 to 28 oC, in different tissues of the white shrimp Litopenaeus vannamei, but some of these antioxidant enzymes also increased when the temperature was decreased from 22 to 15 oC, suggesting a modulation to endure a possible metabolic depression under low temperatures. Similarly, Abele and Puntarulo [72] reported that polar mollusc present higher SOD activity when compared to temperate mollusk, whereas CAT activities were very heterogeneous with high and low activities in both climatic groups.

Indeed, studies have shown that in vertebrates temperature greatly influences the survival of anoxia-tolerant species. Because metabolic rate falls with temperature, animals will survive anoxia longer in the cold. For

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anoxia-tolerant vertebrates, a low temperature means that their glycogen stores last longer; that is, they will survive anoxia as long as they have glucose available. In contrast, for anoxia-intolerant vertebrates, a low temperature means only a slower loss of cellular energy charge and therefore a slower death process [73,74]. Though a similar situation has not been proven in marine invertebrate species, these similarities would be expected, however, further studies must be done to confirm this assumption.

6. Conclusions A deep understanding of antioxidant responses in marine invertebrates under hypometabolic conditions has yet to be achieved. Even the identification of environmental conditions that can cause organisms to enter into a hypometabolic state (i.e. temperature oscillations, food deprivation, salinity and pH variation, etc.) is sometimes difficult to duplicate. As shown with the several marine invertebrates discussed in this chapter, it can generally be assumed that hypometabolic tolerant animals present efficient antioxidant responses to handle ROS that are produced during the hypometabolic state or after the recovery period, or when ideal conditions return in the environment.

However, there is still little understanding of how these antioxidants are modulated over different marine invertebrate species that must respond to metabolic depression, and this topic deserves a special focus in future research.

The knowledge of these mechanisms can aid in the understanding of how such hypometabolic tolerant organisms evolved their adaptative responses to adverse environmental conditions during the course of evolution. This information also contributes to the understanding of the ecological relationships between different species inhabiting severe environments that impose periodic cycles of metabolic depression for animal life.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates, 2011: 57-94 ISBN: 978-81-308-0471-2 Editors: Anna Nowakowska and Michał Caputa

4. Hypometabolism and turtles: Physiological and molecular strategies of anoxic survival Kyle K. Biggar, Amy G. Groom and Kenneth B. Storey Institute of Biochemistry & Department of Biology, Carleton University, 1125 Colonel

By Drive, Ottawa, Ontario, K1S 5B6, Canada

Abstract. A common theme in scientific literature is the association between metabolic rate, energy status and stress resistance. Resistance to environmental stress has long been a focus of comparative physiologists, e.g., research focusing on the environmental extremes of temperature change, water availability and oxygen limitation. Of particular interest is the problem of complete oxygen lack (anoxia), and tolerance to various degrees of oxygen limitation (hypoxia). In some cases, sufficient metabolic depression can be achieved from behavioral or physiological responses allowing the animal to adapt to new oxygen conditions.

However, when conditions are extreme, such as prolonged lack of oxygen, some turtles are able to reorganize cellular functions to facilitate both long-term depression of metabolic rate and survival.

The molecular mechanisms of hypometabolism include global suppression of energy-expensive cell functions (e.g. protein synthesis, gene transcription, ATP-dependent ion pumps), reprioritization of ATP use towards vital cell functions, and enhanced expression of multiple preservation mechanisms Correspondence/Reprint request: Dr. Kenneth B. Storey, Institute of Biochemistry & Department of Biology Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, K1S 5B6, Canada E-mail: kenneth_storey@carleton.ca

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(e.g. antioxidants, chaperones) that protect and stabilize cellular macromolecules. For example, the antioxidant defenses in turtles are constituently high when compared to other ectotherms, and are frequently found to be within the range of endothermic mammals despite the low metabolic rate of cold-blooded turtles. This could suggest that, turtles are ‘preadapted’ to withstand oxidative stress and oxygen reperfusion injuries associated with transitions to/from hypometabolic states.

The problems of anoxia survival are two-fold. The anoxic turtle must initially reduce its metabolic requirements during the period of low oxygen (hypometabolism), and it must also protect itself against oxidative stress during the oxygen reperfusion period that follows. Because turtles routinely experience periods of low oxygen availability, during diving or overwintering, these organisms have provided important insights into the mechanisms that may be necessary to meet these challenging situations.

List of symbols and abbreviations 4EBP ADP AMP ATP CAT cDNA DNA eIF4E ERK F2,6P2 GABA GPX GR GSH GSSG GST GTP H2O2 HIF-1α HSP HUVEC JNK LDH MAPK miRNA mTOR mRNA

4E-binding protein adenosine diphosphate adenosine monophosphate adenosine triphosphate catalase completmentary DNA deoxyribonucleic acid eukaryotic initiation factor 4E extracellular signal-regulated protein kinase fructose-2,6-bisphosphate γ-Aminobutyric acid glutathione peroxidase glutathione reductase glutathione oxidized glutathione glutathione-S-transferase guanosine triphosphate peroxide hypoxia inducible factor 1, α subunit heat shock protein human umbilical vein endothelial cells c-Jun N-terminal kinases lactate dehydrogenase mitogen-activated protein kinase microRNA mammalian target of rapamycin message RNA

Hypometabolism and turtles

NADH NADPH NMDA O2ODDD .

OH p16INK4a PFK PHD PI3K PK PRX Rb RNA .

ROO ROS SOD UTR

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nicotinamide adenine dinucleotide nicotinamide adening dinucleotide phosphate N-methyl D-aspartate superoxide oxygen-dependent degradation domain hydroxyl radical Cyclin-dependent kinase inhibitor 2A phosphofructokinase proline- hydroxylase phosphatidylinositol 3-kinase pyruvate kinase peroxiredoxin retinoblastoma ribonucleic acid peroxide radical reactive oxygen species superoxide dismutase untranslated region

1. Introduction All the oxygen in the present atmosphere is believed to have had a biological origin, and was mostly formed approximately 2,000,000,000 years ago. Oxygen also is believed to be a product of early photosynthesis reactions carried out by primitive green plants and cyanobacteria. It was only at this time that primitive eukaryotes acquired mitochondria, and life forms evolved to use oxygen as the final acceptor in their electron transport pathways [1].

The ability to link oxygen to catabolism, extracting greater amounts of energy per molecule of organic substrate, has driven evolution to expand life into higher complexity and has made the availability of oxygen critical to the survival of many organisms. However, an extreme dependence on oxygen comes at a cost; mammalian organ systems are designed to function under high oxygen content and every effort is made to maintain the oxygen level within a narrow range of operating limits. Situations of hypoxia or anoxia can rapidly lead to severe tissue damage and inevitably death to intolerant organisms.

Living animals are constantly faced with various environmental stresses that challenge normal life, including; oxygen limitation, very low or high temperatures, water limitation and food restriction [2-3]. Of these stresses, oxygen variation in the environment is one that many animals commonly experience. In their northern ranges, turtles can experience drastic changes in

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oxygen supply, arising by either; (1) variations in environmental oxygen availability (e.g. ice-locked ponds and lakes with hypoxic or anoxic water) that deny turtles access to oxygen for extended periods of the time or, (2) by animal behaviors that interrupt the supply of oxygen (e.g. extended periods of breath-hold diving) [4]. Depending on the length and severity, both of these situations decrease oxygen supply and lead to a restriction in oxidative phosphorylation and mitochondrial ATP production [4]. The decrease in ATP production, can quickly lead to a disruption of many ATP-utilizing processes in the cell. The loss of function in ATP-dependent ion channels can be particularly damaging since it disrupts the normal balance between the opposing rates of ATP-dependent ion pumps versus passive ion channels, resulting in a quick loss of membrane potential [5]. In the brain, this loss of membrane potential causes a rapid breakdown of critical transmembrane ion gradients, a rise in intracellular Ca2+ concentrations and a release of excitatory neurotransmitters [6]. It is this release of neurotransmitters and increase of intracellular Ca2+ that trigger multiple dangerous effects including the signaling of programmed cell death (or apoptosis) [7].

Common to many animals, variations in environmental oxygen levels or behavior that disrupts the supply of oxygen, can create situations of oxygen deprivation that must be tolerated. As a result, most animals have developed mechanisms that allow them to compensate for mild or shortterm hypoxia. In mammals, these responses are activated in order to; (1) improve oxygen delivery to tissues and, (2) increase anaerobic ATP production to compensate for the reduced ATP output. Such physiological responses include an increase in hemoglobin unloading of oxygen, an increase in ventilation and lung gas exchange, as well as the release of stored red blood cells from the spleen [8]. Together, these adaptations serve to increase uptake and delivery capacity of oxygen to organs. The normal metabolic response to oxygen deprivation includes an increase in the glycolytic rate, as well as consumption of creatine phosphate reserves [8].

As previously mentioned, in the majority of cases oxygen deprivation can still be extremely damaging despite physiological responses, and given the severity, are often lethal in intolerant animals.

2. Anoxic survival in turtles The mammalian focus on maintaining optimal oxygen supply to organs is not universal throughout all animals. Many vertebrate organisms can live without oxygen for extended periods of time, functioning as facultative anaerobes [9-10]. For example, oxygen limitation is a daily occurrence for many species of turtles which spend much of their lives underwater, either

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diving for food or escaping predation [11-12]. Winter survival for many freshwater turtles is also ensured by underwater brumation, providing an escape from freezing temperatures.

For short-term oxygen deprivation, such as diving for food, anaerobic metabolism can easily meet metabolic demands [8]. However, for long term survival, such as overwintering underwater, turtles turn to one of two modes of survival; (1) extrapulmonary mechanisms of oxygen uptake, and (2) a decrease in metabolic demand (hypometabolism). For most turtles, this capacity to survive underwater without pulmonary ventilation stems in part from their proficiency in exchanging respiratory gases with water across a well vascularized epithelium lining the throat and/or cloaca [8,13]. This method of gas exchange, known as extrapulmonary oxygen uptake, is most often utilized by soft-shelled turtles [14]. For example, many subtropical Australian turtles can utilize extrapulmonary gas exchange to obtain adequate oxygen supply even in warm water (when dissolved O2 is low) [15]. However, other turtles utilize a depressed metabolic rate and lowered oxygen demand to survive winter months underwater, perfecting facultative anaerobiosis.

The best facultative anaerobes among freshwater turtles include members of the genera Trachemys (pond slider turtles) and Chrysemys (painted turtles).

These species are widespread over most of the United States and southern Canada. Sliders and painted turtles are able to survive without oxygen for up to two weeks at ~16째C and for 12 to 18 weeks at ~3째C [13, 16-18].

Comparatively, mammalian tissues are intolerant to even short episodes of anoxia, whereas turtle tissues rapidly decrease their metabolic rates to ~10% of normoxic resting rates, and buffer the lactic acid produced by anaerobic glycolysis in their bony shell [12, 17].

The focus of this chapter comes from the existing research on the biochemical and physiological responses characterizing metabolic rate depression in the turtle. In many species, living with low oxygen is an everyday occurrence; thus, studies of metabolic adaptations which promote survival in these species encompass both biochemical adaptations and physiological responses. On the other hand, any limitation on the supply of oxygen may also be a life-threatening stress in oxygen sensitive cells. In these systems, cell function and structure may become disturbed and irreversibly damaged. The goal of many studies examining anoxia-induced hypometabolism-based research is to identify the mechanisms that can promote survival in these oxygen-sensitive cells and the turtle is an excellent vertebrate model for this.

The emphasis of this chapter is on four fundamental defense strategies which are examined in several model turtle species as they are frequently observed in anoxia tolerant organisms:

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1) The physiological responses to low oxygen and the cellular transition to the anoxic state. This includes mechanisms of signal transduction, and metabolic reorganization.

2) Suppressing energy demand as a means to rebalance ATP homeostasis during anoxia, thus avoiding the negative consequences of energy failure.

3) Minimizing the damage caused by oxygen reperfusion upon reoxygenation after anoxia.

4) Utilizing signaling cascades as a means to rapidly detect extracellular stress and promote survival.

Additional emphasis is given to molecular strategies of hypometabolism that protect anoxia-tolerant turtles (e.g. unfolded protein response, antioxidant defense) and to emerging areas of research in the mechanisms of global metabolic control (MAPK, post-translational modifications, and small non-coding RNA) when oxygen is limited.

2.1. Survival strategies of hypometabolism in hatchling and adult turtles To escape harsh winter conditions, turtles have adapted many different survival strategies. For example, as a means to avoid northern winters, sea turtles are able to migrate vast distances to a warmer climate [19].

Unfortunately, most other turtles do not have this ability and must cope with winter stress in another way. Interestingly, hatchlings of some species of turtles spend their first winter in or below the nest cavity (<10 cm deep, typically on exposed river or lake banks) where temperatures can easily drop below the point of freezing [20]. To survive the extreme drop in body temperatures, hatchling turtles have developed two strategies; (1) allowing their body temperatures drop below the freezing point (-2.10 ¹ 0.21°C) while remaining in an unfrozen state, a process known as freeze-avoidance, and (2) allowing the complete freezing of their tissues (conversion of ~50% of total body water into extracellular ice). Interestingly, it has been shown that freeze tolerant hatchling turtles vary in their ability to survive anoxia [21-24]. This is unexpected as it has been documented that a significant element of freeze tolerance is ischemia/anoxia resistance, resulting from the freezing of blood plasma and halting oxygen delivery to organs. This suggests that ischemia/anoxia is a co-stressor towards freeze tolerance. Whether or not these traits are associated with each other has not been determined. However, large stores of liver glycogen, present in both hatchlings and their adult counterparts, may contribute to the survival of both hatchling and adult turtles during both types of stresses [22]. When freezing initiates, glucose is produced from glycogen stores found in the liver, and is delivered to all the

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organs through circulation [25-26]. It is this glucose that provides protection from desiccation by enhancing the colligative properties of the cell and reducing damage to membrane structure [for review see 26]. Incidentally, glucose is also a fermentable substrate supporting anaerobic production of ATP during oxygen lack [27]. It then could be assumed that species that maintain large glycogen reserves would be naturally predisposed to survive freezing in addition to anoxia survival. Unfortunately, hypometabolic studies on hatchling turtles are only beginning to emerge and the vast majority of research has focused on the anoxia tolerance of adult turtles, and hence will be the focus of this chapter.

Little research has focused on the discrepancy of anoxia tolerance between adult and hatchling turtles. However, some studies have suggested that this divergence in anoxia tolerance arises from the fully ossified shells in adult turtles [21, 28]. The ossified turtle shell acts as a mechanism to buffer anaerobic metabolites, namely lactate, through the release of carbonate minerals into extracellular fluids (Figure 1) [28]. If buffering reserves are not adequate or fully developed, an excessive drop in intracellular pH may ultimately result in death. To highlight this point, several studies have suggested that it is the buildup of lactic acid that contributes to freezing mortality when seen in hatchling turtles, presumably attributed to an underdeveloped shell and a decrease in associated buffer capacity [21-22].

2.2. The hypometabolic response Upon sensing a decrease in oxygen availability, the first response of the anoxia tolerant turtle is to increase oxygen extraction and delivery systems.

These mechanisms are well established in the literature and include the physiological responses of increasing lung ventilation, alterations to hemoglobin oxygen affinity leading to enhanced oxygen extraction, as well as, increasing cardiac output to improve oxygen delivery to organs [29-30]. If oxygen concentrations continue to fall, the systemic alterations to oxygen extraction quickly become inadequate to supply enough oxygen to deprived tissues. Once this occurs, oxygen-independent metabolic pathways, such as anaerobic glycolysis, are fully recruited and are followed by cellular alterations to reduce oxygen demand [29-31]. This introduces a very important issue, increasing the rate of glycolysis does increase ATP output, but also results in a quick depletion of internal carbohydrate fuel reserves, as well as a large accumulation of acidic end products [32-33]. Hypometabolic turtles must therefore cope with this problem. Freshwater turtles, such as the painted turtle (C. picta bellii), accumulate plasma lactate concentrations as high as 150-200 mM after several winter months [34-36]. In comparison, a

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human exercised to exhaustion may only experience an extreme plasma lactate level of 20-25 mM [37]. This acidic load far exceeds the natural buffering capacity of plasma bicarbonate (35-45 mM) and the turtle is able to cope with these extraordinarily high lactate levels by utilizing key physiological resources, namely its calcium carbonate-rich shell and skeleton [35-36]. Primarily, carbonate minerals are dissolved into the extracellular fluid and act to form complexes with lactic acid and supplement buffering (Figure 1). As an additional mechanism, lactic acid is taken up by both the shell and bone, where natural carbonate acts to buffer and store lactate until normoxic conditions are restored [35-36].

When oxygen supplies are completely cut off (anoxia), ATP demand soon outstrips ATP supply and neural cell death is inevitable for intolerant animals. For example, once blood oxygen levels fall below optimal in humans, oxidative production of ATP is halted and ATP levels rapidly fall.

If the decrease in ATP levels is not corrected, an imbalance between ATPdependant ion pumps and passive ion channels is created and as a result, membrane potential difference collapses [38]. Once a collapse of membrane potential has occurred, there is a large influx of Ca2+ through plasma membrane channels and a variety of irreversible destructive events, such as

Figure 1. Lactate movement into a calcium carbonate rich shell during anoxia in the hypometabolic turtle. Shuttling of lactate to the shell during anoxia acts to buffer the acidic influence of lactic acid on intra- and extracellular pH levels. Figure modified from [37].

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apoptosis, are initiated (many of them Ca2+-mediated) [39]. Additionally, increasing ATP supply via anaerobiosis, quickly consumes substrate and inevitably serves only to shorten survival time. Thus, it is the reduction of ATP demand, not an increase in glycolysis that is the only viable long-term strategy for a vertebrate to survive without oxygen.

Turtles escape anoxia-induced death by suppressing, rebalancing and reprioritizing the rates of ATP-utilizing and ATP-generating processes, so that they can sustain long term viability without oxygen [40]. Both ATP production and ATP consumption are strongly decreased in response to anoxia in tolerant turtles. For example, studies with isolated turtle hepatocytes showed a 94% decrease in overall ATP turnover when exposed to anoxic conditions [31]. Dramatic changes were seen in the portion of ATP turnover that was devoted to five main ATP consuming processes: (1) ion motive ATPases, (2) protein synthesis, (3) protein degradation, (4) gluconeogenesis, and (5) urea synthesis. By reorganizing key cellular processes, the turtle can redirect available energy stores to vital processes critical for anoxic survival. This results in the most efficient ATP utilization under energy-limited conditions, and is the main characteristic of hypometabolism in many organisms, including turtles. As reported by Hochachka and colleagues [41], the main features of anoxic survival via hypometabolism include: 1) Oxygen sensing and signal transduction pathways that communicate the hypoxic/anoxic transitions to cells 2) A set of genes that are up-regulated 3) A set of genes that are down-regulated 4) A decreased activity in non-essential pathways 5) A decline in membrane permeability and impulse frequency in neural tissues, and 6) A sustainable balance of ATP utilization and production These are the fundamental, and highly regulated, processes that allow hypometabolic turtles to survive extended periods of reduced oxygen (Figure 2) [41]. Indeed, in turtles, a profound metabolic rate depression to only 10-20% of the corresponding aerobic resting rate, at the same temperature, occurs in response to anoxia [42].

2.3. Transitions in hypometabolism Considerable research on metabolic depression in turtles has defined a number of critical, and highly regulated, transitions to/from the hypometabolic

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state [31, 43]. These transitions include: (1) an initial downregulation of ATP utilizing processes during the transition between hypoxia and anoxia, (2) longterm maintenance at a metabolically depressed state and (3) a rapid upregulation of metabolic rate and normal cellular activity when oxygen becomes available. Previously, we introduced the physiological responses and ATP reprioritization of the first transition phase, entrance into hypometabolism.

This process involves a drastic reduction of ATP use during the first few hours of hypoxia, in response to declining O2 tensions, so that ATP demand can be satisfied through anaerobic glycolysis (Figure 2) [44]. The transition to a hypometabolic state is highly regulated, achieving a suppression of many cellular processes and the re-establishment of ATP homeostasis between ATPproducing and ATP-utilizing reactions. A failure to meet cellular ATP requirements during this period is the difference between long-term survival and catastrophic cell death. Regulation of this entry phase is highly coordinated through rapid post-translational modification of cellular enzymes and signaling pathways [3, 45-46]. However, when arterial O2 tension drops below a critical limit (arterial pO2 of 20 Torr in turtles), a long-term strategy to conserve ATP supply is initiated [47].

The second phase of the hypometabolic transition in turtles is the entry into a maintenance period (Figure 2). This is the longest hypometabolic period in the turtle and can last from hours (long dives) to days or months (overwintering) and involves the maintenance of cellular processes and homeostatis at an order of magnitude lower than the normoxic state. Research in recent years has also started to define mechanisms that regulate an overall strong suppression of transcription and translation, while enhancing the expression of selected genes and proteins with protective function [3, 48-49].

Much of the research to date, focusing on the maintenance of hypometabolism in turtles, has been concerned with energetics, fuel catabolism, and molecular controls on energy-expensive cell functions. These functions include controls on gene transcription, cell cycle, protein translation, and neuronal ion-motive ATPases. It also should be noted that turtles have been found to have constitutively high levels of antioxidant enzymes that are maintained throughout this phase [50]. The activities of these enzymes are much higher than those in other ectothermic vertebrates, and are actually comparable to mammalian activities despite the metabolic rate of turtles being much lower. It has been proposed that a constitutively high antioxidant defense is a result of natural adaptation, or preconditioning, of turtles for the rapid oxygen reperfusion that result upon reoxygenation [50-51].

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The final transition phase is the exit from hypometabolism, once normoxic conditions have been restored (Figure 2). Similar to the transition into hypometabolism, once the anoxic stress has been removed there must be an equally regulated reactivation of suppressed cellular activities and processes. However, transitioning back to the normoxic state is not as simple as reactivating each metabolically depressed process. Within the first 10 min of recovery, blood oxygen rapidly returns to normoxic levels [50]. The reintroduction of oxygen to the turtle brings about a flood of damaging reactive oxygen species (ROS) and protective mechanisms must be put into place. Damage resulting from uncontrolled generation of ROS can lead to peroxidation of polyunsaturated fatty acids in organelles and other plasma membranes. Free radical exposure may also result in the oxidation of sulfhydryl-containing enzymes, carbohydrates (polysaccharide depolymerization) and nucleic acids (single and double strand scissions) [52-53]. To deal with the potential of ROS induced cellular damage, the red-eared slider turtle (T. scripta elegans) maintains high constitutive activities of various antioxidant enzymes including catalase, superoxide dismutase (SOD) and alkyl hydroperoxide reductase [50]. In order to survive each of these transitional phases to and from the hypometabolic state, the turtle successfully negotiates the separate requirements needed at each step. The hypometablic transition requires a highly controlled regulation at both the physiological and molecular levels.

Figure 2. Transitions to and from a hypometabolic state in the anoxic turtle. Upon initial hypoxic sensing, cellular adjustments occur to reprioritize ATP metabolism and defend the cell from oxidative damage upon oxygen reperfusion. Figure modified from [41].

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2.4. Hypometabolism in the turtle brain In intolerant animals, such as humans, entry into a hypoxic (or ischemic) state results in the loss of neuronal membrane potential, leading to large releases of excitatory neurotransmitters (such as glutamate) and neurotoxicity [43, 54]. Interestingly, this course of events does not occur in the hypometabolic turtle. The mechanisms of anoxia tolerance in the turtle brain include ion channel arrest, increase in active uptake of glutamate (an excitatory neurotransmitter) and the increase of both Îł-aminobutyric acid (GABA) release (an inhibitory neurotransmitter) and GABAA receptor amount [43, 55-56]. In general, these mechanisms protect the turtle brain against anoxia and effectively silence much of the brain activity throughout the prolonged hypometabolic state.

In 1986, Hochachka made the first suggestion that an arrest of ion channel activity plays a critical role in maintaining ion homeostasis in the turtle brain throughout hypometabolism [57]. The energetic requirements of neuronal ion channel pumping accounts for ~50% of the energy used by the normoxic neuron [38]. Therefore, the reduction (or arrest) of ion channel activity, or ion permeability, would contribute significantly to the overall reduction of ATP demand during hypometabolism. Several studies by the Lutz lab, have documented the use of this strategy in the anoxic turtle brain [58]. Although the permeability of ions through the plasma membrane in turtle is already greatly reduced, a further decrease in ion channel activity and channel number during anoxic exposure has been suggested. Indications of ion channel arrest include the maintenance of membrane potential contributed by; (1) a decrease in Na+-K+ ATPase activity by 75% in turtle hepatocytes, (2) an anoxia-mediated 42% decrease in voltage-gated Na+ channel density in turtle cerebellum, and (3) a 62% decrease in N-methyl D-aspartate (NMDA) channel open time in the turtle cerebrocortex [58-59]. In particular, the anoxic regulation of the NMDA receptor during hypometabolism has been researched extensively in the turtle [60]. The regulation of this receptor is likely critical, as this ligand-gated channel is highly permeable to Ca2+ and undergoes regulation of its activity through rapid post-translational phosphorylation.

Lutz et al. has reported that glutamate release and uptake in the metabolically depressed turtle brain is extremely coordinated, maintaining a balance between glutamate release and active uptake mechanisms [61]. It is the massive release of glutamate that is thought to be primarily responsible for the destructive neuronal events, triggering influx of Ca2+ ions and cell death in intolerant animals [62]. During hypometabolism, the anoxic turtle brain displays a controlled reduction of extracellular glutamate release and

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continued operation of glutamate uptake transporters. The reduction of extracellular glutamate levels is further emphasized with the naturally low number of δ-opioid receptors in the turtle cortex when compared to the rat which, displays a four-fold higher concentration of the receptor [63]. This may be an indication that turtle neurons are naturally protected from high levels of glutamate, preventing neurotoxicity during hypometabolism.

In addition to controlled regulation of the excitatory neurotransmitter, glutamate, the turtle also protects itself from neurotoxicity by increasing inhibitory tone (reducing the chance of an excitatory event), effectively silencing much of the brainâ&#x20AC;&#x2122;s activity. Extracellular concentrations of the major inhibitory neurotransmitter, GABA, have been found to reach 80-fold higher than seen in normoxia and begin to increase as early as 2 hours into the anoxic condition [56]. This increase of GABA is facilitated with the increase of GABAA receptor density, strengthening the effectiveness of the GABA and entering the turtle brain into a silenced state [56].

2.5. Satisfying ATP homeostasis with hypometabolism Organisms utilizing aerobic metabolism are able to make use of carbohydrates, lipids, and proteins as oxidative fuels. However, when oxygen becomes limiting, organisms are restricted to the use of carbohydrates as the major fermentable fuel. This means that the major source of energy storage for many animals, lipids, become unusable when oxygen levels fall. Since ATP generation from glycolysis is extremely low (a net of only 2 mol ATP per mol of glucose catabolized), it is clear that ATP supply would soon fall short of ATP demand in a very short period. In order to maintain normal levels of ATP generation (aerobic ATP generation yields 36 mol ATP per mol of glucose catabolized), organisms would have to burn 18-times more glucose than would be normally necessary. This would have deleterious consequences for intolerant animals, as glucose stores would essentially be exhausted and its fermentation would generate high accumulation of acidic waste products (lactate ions and associated protons) [30]. Successful facultative anaerobes, such as turtles, must put in place particular adaptations that allow them to survive the negative consequences of running anaerobic glycolysis. Such mechanisms include: (1) increasing reserves of fermentable fuels (glycogen), particularly in the liver, (2) a tolerance for large changes in the pH of both intra- and extra-cellular fluids and (3) a means to depress metabolic demand such that ATP supply can be adequately matched with low levels of glycolysis. During the early hours of the hypoxic transition, creatine phosphate reserves, in combination with glycolysis, contribute substantially to ATP needs; however, these reserves are quickly

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depleted and are only able to contribute within the early hours of hypoxia and alternative mechanisms for ATP homeostasis must be put into place [11, 65].

3. Detection of extracellular stress As previously eluded, one of the most widely researched mechanisms of metabolic rate depression is the control of protein/enzyme activity via reversible protein phosphorylation [3]. This mechanism reappears across phylogeny as the means of making major changes in metabolic rate and reorganizing metabolism in numerous animal models, including estivating snails (Otala lactea), frozen frogs (Rana sylvatica), anoxic turtles (T. scripta elegans) and hibernating squirrels (Spermophilus tridecemlineatus) [45, 66-68].

Numerous studies have documented the role of reversible phosphorylation in modifying the activity states of regulatory enzymes involved in both oxidative and anaerobic carbohydrate catabolism. In T. scripta elegans the posttranslational phosphorylation of glycolytic enzymes has been linked to the regulation of glycolytic rate (altering the activities of PK and PFK under anoxia), as well as redirecting the carbon flow into catabolic pathways of energy production [44]. Apart from enzymatic regulation, reversible phosphorylation has been documented as a powerful and widespread means of regulating many functional proteins, including transcription factor activity and associated gene expression, rates of ion channel transport across plasma membranes, the state of cellular proliferation, and rapid controls of translational rates [3, 69-70]. As such, it has been proposed that the phosphorylation of functional proteins may be a rapid and coordinated means of readjusting metabolic processes and depressing nonessential cellular processes throughout hypometabolism in anoxic turtles.

3.1. Protein regulation via post-translational modification Through the use of radiolabeled 32P, one study documented an increase in global phosphorylated protein amount during anoxic exposure in T. scripta elegans [45]. Protein phosphorylation patterns during anoxia revealed 1.6-, 2.4-, and 1.3-fold increases in 32P incorporation in anoxic brain, heart, and liver tissues respectively. Reversible phosphorylation control over the activity of the central pathway of carbohydrate catabolism, glycolysis, is one of the most critical features of metabolic depression in many systems, and has been extensively characterized in turtles. The examination of the phosphorylated state of glycogen phosphorylase, PFK, and PK in the turtle showed the influence of phosphorylation on kinetic properties [44]. These organ-specific changes were consistent with anoxia-induced posttranslational modification of the enzymes.

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Apart from direct regulation of functional proteins, reversible phosphorylation is also responsible for the detection of extracellular stimuli through signal transduction networks. The intracellular signaling of anoxia in turtles has focused on the differential regulation of the mitogen-activated protein kinase (MAPK) superfamily. The phosphorylation of a MAPK family member, results in a conformational change in protein structure and a >1000fold increase in kinase activity [71]. In effect, MAPKs are not functional as signaling molecules until phosphorylated by their respective upstream kinases. Once activated, MAPKs phosphorylate their respective downstream proteins, many of which are transcription factors that have key roles in the up-regulation of genes critical to the anoxic stress response [72]. The three main MAPK family members, and their regulation in the hypometabolic turtle, are briefly summarized in the section below.

3.2. Mitogen-activated protein kinase signaling The activity of many intracellular proteins, and their appropriate cellular functions, are regulated by the transmission of extracellular signals, mediated

by MAPK proteins (Figure 3). It has been well documented that these MAPK pathways are key in regulating stress responses and transducing extracellular signals to cytoplasmic and nuclear effectors, and several studies have documented their role in hypometabolic turtles [72-74]. The MAPK superfamily consists of three main protein kinase families: extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and, p38 [72]. MAPK cascades detect, amplify and integrate diverse external signals to generate survival responses, such as changes in protein activity or gene expression [3, 72]. In the turtle, the activation of MAPK signaling may provide a conduit for a rapid response in stress-responsive gene expression, contributing to the turtleâ&#x20AC;&#x2122;s ability to enter a state of anoxia-induced hypometabolism.

Studies on the MAPK activation in anoxic adult and hatchling T. scripta elegans have identified one common result; both ERK and p38 have little or no involvement in the hypometabolic responses of anoxia in turtles [46]. Apart from this finding, the activity of JNK increased in tissues of both hatchling and adult turtles in response to anoxia. In T. scripta elegans, JNK showed maximum activation after 5 hours of anoxia but quickly decreased with increased exposure. This result suggests that JNK may have a role in the hypoxia transition period leading into full anoxia, with JNK suppressed back to control values when a complete depression of metabolic rate has been achieved. It has been suggested that the lack of involvement from the ERK pathway could be a result of its primary response to growth factors [75]. Growth factors are responsible for stimulating

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Figure 3. Generalized signalling pathways of ERK, SAPK/JNK, and p38 including their influences on cell functions.

cell growth, proliferation, and cell differentiation, processes which would lead to a rapid depletion of cellular ATP supply, and inevitably, cell death.

The unchanged activation of p38 in the anoxic turtle is of particular interest as it contrasts with models of anoxia intolerance. It has been shown that by transiently activating the p38 pathway, through short preconditioning exposures to ischemia in the mammalian heart, the recovery of function during reperfusion is significantly improved [76]. Additionally, activation of both JNK and p38 has been correlated with improved survival of in ischemiareperfusion in mammalian kidney [77]. It has been speculated that the metabolic responses to anoxia between tolerant and intolerant organisms may be mediated through the differential regulation seen in the p38 signal transduction pathway; albeit more research is necessary to reach such a conclusion [72]. The distinct patterns of MAPK signaling in the anoxiatolerant turtle, is suggestive of the relative contribution of each signaling pathway in altering cell function and establishing a hypometabolic state.

4. Hypometabolic response to oxygen limitation: Molecular regulation and metabolic organization 4.1. Metabolic reprioritization Control of anaerobic glycolysis has received extensive research in the turtle, partially due to the desire of researchers to understand the molecular

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basis of the Pasteur Effect in tolerant organisms. The initial work studying the effects of oxygen deprivation on metabolism in turtles, focused on the allosteric regulation of enzymes involved in glucose metabolism. These studies had a particular focus on the control of 6-phosphofructokinase (PFK-1) which was thought to be the central and rate-limited enzyme in glycolysis (Figure 4) [78].

The PFK-1 enzyme is highly sensitive to substrate inhibition by high levels of ATP and to allosteric activation by adenosine 5â&#x20AC;&#x2122;-monophosphate (AMP). When the energetic demand for ATP outweighs the ability to supply ATP, the relative levels of cellular ATP soon drop. As an important note, due to the near equilibrium of the adenylate kinase reaction (2 ADP Ă&#x2020; ATP + AMP), a small change in ATP levels translates into a several-fold increase in AMP levels, a PFK-1 activator. Another potent regulator of PFK-1 is fructose-2,6bisphosphate (F2,6P2) [8]. During anoxia in yeast, F2,6P2 has been found to rise and activate PFK-1, facilitating a rise in the rate of glycolysis and an increase in carbohydrate-based fuels, highlighting the Pasteur Effect under anoxia as influenced by F2,6P2 and PFK-1.

Studies in the hypometabolic turtle, T. scripta elegans, have clearly indicated glycolytic activation after 1 hour of submergence from the characterization of PFK-1 enzyme kinetics [32, 44]. After one hour of submergence (early hypoxia), activation of glycolysis was seen in brain, heart and skeletal muscle [32]. However, these same organs showed clear evidence that the previous glycolytic activation was reversed after five hours of submergence. The depression of glycolytic rate seen after five hours of submergence is reflective of the overall depression of metabolic rate and ATP requirements that accompany long-term hypometabolism in turtles.

Interestingly, this same study identified differential regulation of glycolysis in liver tissue, indicating a very rapid glycolytic inhibition occurring within the first hour of submergence. The inhibition of liver glycolysis, facilitated by a reduction of liver F2,6P2 levels, allows glycogenolysis to be directed towards the exportation of fermentable fuel to other organs and satisfy substrate needs [32]. As a result, the available glycogen in turtle organs (brain, heart and skeletal muscle) is utilized for endogenous fermentation within the very early hours of anoxia, whereas long term anaerobiosis is fueled by exogenous glucose supplied from glycogen stores in the liver [11, 32]. In addition, the regulation of glycolysis can also be influenced through the upregulation of rate-limiting enzymes/proteins, such as PFK-1 and PK, by a hypoxia-sensitive transcription factor [79]. This up-regulation of rate-limiting enzymes results from the post-translational stabilization and subsequent activation of the hypoxia-inducible transcription factor HIF-1; this transcription factor is believed to play critical roles in the hypometabolic transition in the turtle.

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Figure 4. Glycolysis in the anoxic turtle. Activation of this pathway occurs upon initial sensing of oxygen lack, but is quickly depressed as part of the transition into a hypometabolic state.

4.2. The hypoxia response: Hypoxia inducible factor-1α The hypoxia inducible factor (HIF-1) transcription factor responds to low oxygen levels and plays an important role in protecting tissues from hypoxia related damage (Figure 5). This protection role includes the up-regulation of selected genes during hypoxia, including those required to improve oxygen delivery to tissues and enhance the capacity of anaerobic glycolysis [80].

HIF-1 is a heterodimer consisting of two subunits: HIF-1α and HIF-1β. HIF-1α and contains a specific oxygen-sensitive region; called the oxygen-dependent degradation domain (ODDD) [81]. This structure is hydroxylated by proline hydroxylase-2 (PHD-2) and degraded by proteosomes, effectively decreasing HIF-1α protein expression under nomoxic cellular conditions [82].

The regulation of HIF-1α expression, like many other genes, occurs on multiple levels including mRNA expression, protein expression, nuclear localization, and trans-activation [81]. However, unique from most proteins, HIF-1 undergoes additional regulation by molecular oxygen (O2). Under normoxic conditions the proline residues, Pro402 and Pro564, found in the ODDD of HIF-1α, are hydroxylated by oxygen-dependent PHD-2 and as a result tagged for degradation [83]. The hydroxylated amino acids in the

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transactivation domains are unable to interact with translational co-activators, preventing the transcription of target genes [81]. However, under hypoxic conditions (O2 concentrations of less than 6% - corresponding to a partial pressure of 40 Torr at sea level), the α and β units are able to bind together and become transcriptionally active [84]. The active HIF-1 complex promotes the transcription of several proteins that are required for anaerobic glycolysis including pyruvate kinase (PK), lactate dehydrogenase (LDH), phosphofructokinase (PFK-1) and pyruvate dehydrogenase kinase (PDK) [83]. An upregulation of PDK by HIF-1 acts to increase anaerobic glycolysis by inhibiting pyruvate dehydrogenase (PDH), reducing pyruvate entry into mitochondria under hypoxic conditions and limiting both the rate of oxidative phosphorylation and generation of ROS [83].

A recent study from our lab has characterized some elements of the HIF-1α response in anoxic turtles (T. scripta elegans) and have shown an increase in HIF-1α total cellular protein and HIF-1α nuclear localization in heart during anoxic submergence (Figure 6; unpublished results). The overall amount of HIF-1α was found to increase 2.4-fold compared to the control value, in heart

Figure 5. Hypoxia inducible factor (HIF-1) activation. During normoxia, hydroxylation of the HIF-1α subunit leads to polyubiquitination and proteasome degradation. Under hypoxic conditions, hydroxylation of the HIF-1α subunit is inhibited. As a result, HIF-1α escapes degradation, binds to the HIF-1β subunit and activates the transcription of hypoxia-sensitive genes.

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Figure 6. Effect of 20 hours of anoxic submergence at 4°C on HIF-1α protein in heart of T. scripta elegans: total protein levels and distribution between nuclear and cytoplasmic factions. Details of animal experiments are as in [117]. Histogram shows the ratio of normalized mean band intensity (± s.e.m., n = 4) for normoxic (control) versus 20 hour anoxic turtles. * – values for anoxia are significantly different from normoxic control values (P <0.05).

after 20 hours of anoxia, while nuclear abundance of HIF-1α also increased 2-fold and a decrease of cytoplamsic HIF-1α levels was also observed. Thus, it appears that HIF-1α activity in T. scripta elegans may be controlled by altering the overall amounts of protein, but also by relocalization of the protein to different parts of the cell. In this regard, post-translational modifications are probably important in controlling HIF-1 activity. In conclusion, a role for HIF-1α in T. scripta elegans anoxia tolerance during the early transition into anoxia is strongly suggested from the results of these experiments. Based purely on the energetic needs of heart tissue, differential regulation of HIF-1α abundance seen in the heart, in combination with nuclear localization, implies that HIF-1 may play a critical role in triggering an upregulation of protein synthesis for glycolytic enzymes.

5. Oxygen reperfusion injury: Defense strategies in the turtle 5.1. Generation of free radicals Despite the overwhelming idea that oxygen acts solely as the “good guy” in aerobic metabolism, there are cases when oxygen is a source of DNA, protein, and lipid damage. Oxygen, as a gas molecule, contains two unpaired

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valence electrons in parallel spins, restricting it to only accept one electron at a time when oxidizing a molecule. Numerous biological donors can facilitate the incomplete reduction of oxygen, leading to superoxide formation (O2-).

Such donors include soluble oxidases (e.g. xanthine oxidase), and ubiquinone and NADH dehydrogenase located in the mitochondrial electron transport chain [51]. Hence, the generation of O2- can be coupled with metabolic rate.

Superoxide is not particularly reactive in and of itself, but can inactivate enzymes, cause signal and double strand DNA breakage or initiate lipid peroxidation (membrane damage) if allowed to become reduced to its hydroxyl radical (.OH) form [87]. Under normoxic conditions, about 1-4% of all O2 consumed by mammalian mitochondria is converted to O2- as a result of a leaky mitochondrial electron transport chain [51, 88]. The generation of O2- under normal metabolic activity is not an issue, as it is involved in normal signaling pathways. However, under environmental stresses or rapid reintroduction of oxygen, excess O2- and hydrogen peroxide (H2O2) formation can increase dramatically and induce damaging effects to cell structure [89]. If extensive damage is caused to the mitochondria, the cell signals an activation of apoptosis [90].

As previously mentioned, several turtles are able to survive months of oxygen deprivation while overwintering by severely depressing their metabolic rate. Upon recovery from anoxia, glycolytic ATP production is replaced by ATP production by oxidative phosphorylation. However, during oxygen deprivation, the electron carriers of the electron transport chain become reduced [51]. The reintroduction of oxygen brings about an immediate oxidation of these carriers and an overproduction of O2- and other reactive oxygen species (ROS). This burst of free radical production can overwhelm the antioxidant defenses of intolerant systems. Interestingly, the turtle can tolerate such periods of high oxidative stress as they exhibit a characteristically high level of antioxidant defense when compared to similar ectothermic animals, and display comparable levels to endothermic mammals [51]. The reperfusion of oxygenated blood to ischemic organs happens in parallel with an overgeneration of ROS (mostly formed by mitochondrial respiration) and induction of lipid peroxidation, protein oxidation and DNA damage [91]. This is analogous to the well-studied situation of oxidative stress in mammalian organs subjected to ischemia and reperfusion events such as heart attack and stroke [88]. The fundamental difference between the reoxygenation of turtle tissues after months of anoxia, and the human heart immediately after an ischemic event, is that turtles have a well-developed and constituently high level of antioxidant defense effectively protecting cellular macromolecules against the oxidative stress of reperfusion.

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5.2. Defense against free radicals Damage to critical macromolecules may be far removed from the initial site of radical reaction. For example, free radical production from mitochondrial processes may lead to peroxidation of polyunsaturated fatty acids in other organelles and plasma membranes [52-53]. Damage may also occur to carbohydrates (polysaccharide depolymerization) and nucleic acids (single and double strand scissions) [53]. Free radical damage during perfusion with reoxygenated blood is frequently termed post-anoxic 'reperfusion injury' and is commonly seen during organ transplant and the destruction of coronary artery obstruction [92]. Prevention of ischemic reperfusion injury, upon exit from hypometabolism in anoxia tolerant turtles, is currently attracting much research. If clinically applicable, anoxicreperfusion studies in turtles may mitigate the effects of free radical damage in heart and stroke patients.

Reactive oxygen species include hydrogen peroxide (H2O2), the superoxide anion radical (O2-), the hydroxyl radical (.OH) and the peroxide radical (.ROO). Of these, .OH is the most highly reactive and the least specific in the type of molecules it damages [93]. The hydroxyl radical may be produced from H2O2 through the Fenton reaction or from H2O2 and O2- through the Haber-Weiss reaction (Figure 7). All animals have enzymatic

Figure 7. General mechanisms showing the various biochemical processes of several antioxidant defence pathways.

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mechanisms to protect against reactive oxygen species. Key enzymatic players in the defense mechanism against ROS include catalase (a peroxisomal enzyme that plays a major role in the decomposition of H2O2 forming H2O and O2), superoxide dismutases (Mn-SOD, mitochondrial isoform and CuZn-SOD, cytosolic isoform), glutathione (GSH) and glutathione S-transferases (GST) (Figure 7) [51, 88]. There are also several auxiliary enzymes that are involved in the antioxidant defense system. These include glutathione reductase (GR) which, acts to restore GSH activity back from the oxidized form of glutathione (GSSG), and GPX which reduces free hydrogen peroxide to water and oxygen (Figure 7).

GSH synthetase is another key auxiliary enzyme involved in the antioxidant defense, as it is the enzyme responsible for the formation of GSH [51, 88].

Additionally, at high concentrations, H2O2 can be removed by catalase (CAT). Organisms may make pre-emptive changes in their antioxidant defenses during the anoxic period; this allows these organisms to deal with a burst of oxygen free radical generation during the recovery stage of hypometabolism [50, 51].

5.3. Antioxidant defenses in the turtle Initial studies looking at the antioxidant capacity of turtles focused on the freshwater South American turtle, Phrynops hilarri, which overwinters underwater [94-95]. The latter of these studies proposed that sulfhydryl-rich hemoglobins could quench oxyradicals that are formed during reoxygenation [95]. Although the study contained no systematic analysis of the antioxidant defense system, it did provide brief insight into the antioxidant defenses in the turtle. Several years following, our lab explored many of the molecular aspects of the antioxidant defense in the freshwater red-eared slider turtle, T. scripta elegans, and hatchling painted turtles, C. picta marginata [50, 91, 96]. Two of these studies examined the activities of antioxidant enzymes both during a state of anoxic metabolic rate depression and oxygen reperfusion upon exit from hypometabolism [50, 96].

Maintenance of high levels of antioxidant defenses can prevent oxidative damage from successive bouts of anoxia and recovery. In T. scripta elegans, the tissue pools of glutathione and levels of ascorbic acid (an organic compound with antioxidant properties) have been found to be higher in turtle organs compared to other ectotherms [96,97]. One pertinent study explored the activities of several antioxidant enzymes, in addition to the auxiliary enzyme, GSH synthetase [50]. Interestingly, exposure of T. scripta elegans to long-term anoxia (20 hours) brought about a decrease in select enzyme activities in various tissues (Table 1). The enzyme activity of catalase, GR

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Table 1. Activities of catalase (CAT), superoxide dismutase (SOD), glutathione synthetase (GSH-synthetase), glutathione reductase (GR), and glutathione S-transferase (GST) in six organs of turtles, T. scripta elegans, under normoxic (control), 20 hour anoxic, and 4 hour aerobic recovery conditions. Data are reported as means (± s.e.m., n = 4). ‘a’ – values are significantly different from normoxic control values (P <0.05). Data take from [50] and [96].

and GST showed a reduction of 31-67% in anoxic heart, while the activity of SOD was reduced by 15-30% in the brain and liver. Additionally, the enzyme activity of catalase decreased by 80% and 68% in both brain and kidney tissues respectively, however, GR activity increased by 52% in anoxic liver [50]. Although many of these reductions in enzyme activity may reflect a reduced potential for oxidative damage in the metabolically depressed and anoxic state, many of these changes were reversed after 24 hours of aerobic recovery (reoxygenation) [50]. Both SOD and GR increased by 45 and 64% in turtle heart after 24 hours of aerobic recovery, while GSH synthetase activity doubled in the brain. Combined, these antioxidant defense mechanisms could help the turtle avoid oxidative damage during situations of oxygen variability. It has been shown that the ratio of GSH/GSSG, which decreases under oxidative stress in intolerant organisms, actually increases during recovery from anoxia exposure in turtles. This suggests that no

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oxidative stress occurs during reoxygenation [96]. In addition, oxidative damage products (lipid peroxidation) were largely unaffected over the course of anoxia/recovery in turtle organs [96].

Apart from direct measurements of enzyme activity, the use of cDNA array screening has identified several iron storage and antioxidant genes that are up-regulated by anoxia exposure in the heart and liver of hatchling painted turtles, C. picta marginata [98]. Both the heart and liver showed an increased expression of the heavy and light chains of the iron storage protein, ferritin. The presence of free iron in the ferrous state (Fe2+) can contribute to the state of oxidative stress by participating in the Fenton reaction, with H2O2, to generate highly reactive hydroxyl radicals (.OH) [99]. Perhaps by increasing the abundance of ferritin, a large protein (450 kDa) capable of surrounding a core of 4500 iron atoms in a low reactivity ferrihydrite state, intracellular free iron levels are kept low, minimizing hydroxyl radical production [100]. Additionally, array screening has identified several other antioxidant enzymes that show increased transcript levels in response to anoxia in C. picta marginata including: SOD-1, glutathione peroxidase (GPX) isozymes 1 and 4, GST isozymes M5 and A2, and peroxiredoxin 1 (PRX) [98]. Also, several studies have evaluated the activities oxidative defense enzymes in several species of hatchling turtles. The activity of Îł-glutamyltranspeptidase, an antioxidant enzyme involved in glutathione metabolism, increased 1.8 fold during thawing/reoxygenation after freezing in liver of C. picta marginata [101]. Additionally, catalase activity increased 3-4 fold under both freezing and anoxia exposure in livers of several hatchling turtles; C. picta marginata, T. scripta elegans and Chelydra serpentina [22].

6. Suppression of protein translation 6.1. Translational suppression The suppression of protein synthesis during hypometabolism is vital to anoxic survival in turtles. Protein synthesis consumes a substantial portion of available ATP turnover under normoxic conditions, using about 5 ATP equivalents per peptide bond formed and the synthesis of proteins is well known to be sensitive to the availability of ATP [102]. Appropriately, some freshwater turtles have been shown to decrease the rate of ATP utilized by protein synthesis to only ~6% during anoxia [31]. In this manner, the suppression of protein translation appears to be a protective response to metabolic rate depression in response to environmental stressors, such as anoxia. Several studies have explored the in vivo protein synthesis rates

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during anoxia-induced metabolic depression in turtles [6, 45 and 103]. Fraser and colleagues stated that the rates of protein synthesis in several tissues of T. scripta elegans exposed to 1 hour of anoxia, showed no significant changes from control values [6]. However, these rates soon decreased to ~0% (below measurable values) when the duration of anoxia was increased to 3 hours at 23°C. These results are comparable to those obtained from isolated T. scripta elegans hearts [104-105]. Additionally, experiments using isolated hepatocytes from C. picta bellii, documented a reduction to only 8% of normoxic protein synthesis rates after 12 hours of anoxia [103] By evaluating translational rates after exposure to anoxia, it has been shown that both T. scripta elegans and C. picta bellii successfully suppress protein synthesis without the generation of a ‘protein debt’ [6]. Rates of translation were shown to be unchanged from normoxic values in T. scripta elegans tissues after 3 hours recovery from 3 hours of anoxic exposure [6].

Again, these results are comparable to those obtained from isolated T. scripta elegans hearts after 1 hour recovery from 2 hours of anoxia [106]. Isolated hepatocytes from C. picta bellii exhibited a significant increase of 160% after 1 hour of recovery from 12 hours of anoxia, however, rates of synthesis decreased to normoxic levels after 2 hours of recovery [103]. The initial increased rate of synthesis seen in hepatocytes from C. picta bellii could be a result of a longer anoxic exposure time (12 hours compared to 2 hours anoxia for all other reported studies) or a result of the different environmental conditions pertaining to cells in culture and those in functioning organ systems. In conclusion, upon exposure to short turn anoxia (less than one hour) both T. scripta elegans and C. picta bellii show no decrease in protein synthesis. This result is expected as turtles would still be depending on existing oxygen reserves at this time. However, exposure to 3-12 hours of anoxia, both T. scripta elegans and C. picta bellii display a complete suppression of protein synthesis with little to no protein dept upon restoration of aerobic metabolism, perhaps dependant on the length of anoxic exposure.

6.2. Extracellular control of translation via PI3-K/Akt signaling Inhibition of protein translation during hypometabolism can be achieved in two ways; (1) through the reduction of mRNA substrate and (2) through the differential regulation of ribosomal translational machinery. Due to the high cost of transcription (often ~10% of ATP turnover during normoxia) one would expect to see a downregulation of RNA synthesis under anoxia, leading to the reduction in protein synthesis. However, neither total mRNA content nor the specific mRNA transcript levels of most constitutively expressed genes in the hypometabolic turtle are actually depressed [107]. For

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example, we used complementary DNA (cDNA) array screening in liver tissue from adult T. scripta elegans (control vs. 5 hours anoxia) and hatchling C. picta marginata (control vs. 4 hours anoxia and 5 hours freezing) turtles to assess changes in gene expression during hypometabolism. Regardless of the type of environmental stress (anoxia or freezing) both species of turtle showed that 93-95% of the genes examined were unchanged in transcript levels. A putative up-regulation of 3% of total genes, and a down-regulation of 4%, was seen for T. scripta elegans liver tissue after 5 hours of anoxia.

Liver of hatchling C. picta marginata showed an up-regulation of 2% of total genes after exposure to either 4h anoxia or 5 hours of freezing and a downregulation of 5 and 3% of genes in response to these stresses, respectively (unpublished data).

Similarly, other studies have shown that the RNA-to-protein ratio does not significantly change in T. scripta elegans liver after 12 hours of anoxia [103]. Additionally, complementary studies have documented no change in total translatable RNA concentrations after 16 hours of anoxia or recovery in the liver, kidney, heart and red and white skeletal muscle of T. scripta elegans [107]. Hence, the decrease in protein synthesis rates exhibited during anoxia does not appear to be controlled by tissue RNA concentration. Instead, reversible control of the rate of protein synthesis in response to metabolic rate depression could be invested in the control of ribosome assembly.

Control of protein translation can also be established through the regulation of ribosomal translational machinery. This control can be implemented through the PI3-K/Akt signaling pathway. As a major signaling protein kinase, Akt is involved in the translational rate, through the regulatory phosphorylation of the eukaryotic initiation factor 4E (eIF4E) [108-109].

Regulation of this initiation factor influences the recruitment of other factors that are critical for ribosome binding [110]. Apart from the direct influence on ribosome assembly, the PI3-K/Akt signaling pathway can act to activate another key regulator of translational rate, mammalian target of rapamycin (mTOR). Once active, mTOR is able to phosphorylate the 4E-binding protein 1 (4E-BP1), leading to the release of 4E-BP1 from its inhibitory interaction with eIF4E. The release of eIF4E allows for the initiation of ribosomal biogenesis and protein translation [111]. As such, there are both direct (activation of eIF4E) and indirect (inactivation of 4E-BP1) interactions between PI3-K/Akt and the activation of eIF4E which present the critical link between PI3-K/Akt activation and translational rate. Although studies have yet not explored the regulation of the Akt pathway in the turtle, research in this area may yield interesting promise in revealing potential mechanisms of reversible translational repression.

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6.3. The unfolded protein response: Heat shock proteins Although the global rate of protein synthesis decreases in the turtle upon entry into anoxia-induced hypometabolism, many proteins with key roles in organismal survival are up-regulated during this period [3]. Unfortunately, many of these proteins may be particularly sensitive to the intracellular changes in pH and redox state, cytosolic conditions that are naturally changed during anoxia and reoxygenation. Under these conditions, proteins can lose their native folded conformation to become misfolded and inactive.

Upregulation of heat shock proteins (HSPs) is one of the best known cytoprotective mechanisms, aiding in the expression of key survival proteins, in response to stress (Figure 8) [112]. Most HSPs act as chaperones, helping to fold newly translated proteins, as well as aiding in the refolding of misfolded proteins under stress conditions and signaling the degradation of unstable proteins [112-113]. By their chaperone action, HSPs help to preserve cellular proteins and extend their functional life.

Several studies have explored the cellular expression of HSPs during anoxia-induced metabolic depression in turtles [114-117]. One study found significantly higher levels of nuclear localized HSF1, the transcription factor responsible for HSP expression; levels increased significantly in the heart (2.7 ± 0.5-fold), liver (1.6 ± 0.2-fold), kidney (1.6 ± 0.1-fold) and skeletal muscle (1.8 ± 0.1-fold) in 20 hour anoxic T. scripta elegans [117].

Transcription factors must migrate to the nucleus to exert their effect and hence, changes in the amount of active HSF1 in the nucleus is a key indicator of the state of HSP gene expression. This same study examined the state of HSP protein expression (including Hsp25, Hsp40, Hsp60, Hsp70 and Hsp90) in the heart, liver, kidney and skeletal muscle tissues in 20 hour anoxic T. scripta elegans. Of particular interest was the up-regulation of several of these proteins in liver (Hsp40, Hsp60, and Hsp70), kidney (Hsp25, Hsp40, and Hsp90) and skeletal muscle (Hsp25, Hsp40, Hsp70 and Hsp90) tissues, while no significant up-regulation of HSPs was found in anoxic turtle heart. One additional study identified differential expression of Hsp60 in the heart of anoxic C. picta marginata turtles, compared to anoxia intolerant soft-shelled turtles, rabbits and rats [118]. Hsp60 is a predominantly mitochondrial chaperone involved in the folding of proteins entering the mitochondria. Hsp60 also has protective effects against oxidative stress [119]. As in the case of antioxidant proteins, HSPs and other molecular chaperones also show an upregulation in response to anoxia in turtle tissues. Activation of the heat shock response during anoxia might help maintain protein stability as well as serve as a preparative mechanism for re-oxygenation, since increased HSP expression might also actively prevent damage following oxidative stress.

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Figure 8. Heat shock protein (HSP) activation. During periods of cellular stress, re-folding of unfolded or misfolded protein may be assisted by HSPs. Depending on the extent of cellular stress and erroneous folding, proteins may be stored in aggresomes or undergo proteasomal degradation.

7. Future studies 7.1. Cellular regulation via non-coding RNAs MicroRNAs (miRNAs) are short, non-coding RNAs capable of regulating protein expression within a cell (Figure 9). These 18-25 nucleotide transcripts are able to bind with full or partial complementarity usually to the 3â&#x20AC;&#x2122; untranslated regions (UTR) of mRNA targets, resulting in the inhibition of translation or degradation of that target [120-122]. It is estimated that at least 60% and up to 90% of all mammalian mRNAs may be targeted by miRNAs [123-124], and at this time over 1400 miRNAs have been identified in the human genome. A single miRNA may target multiple mRNAs, and a single mRNA may have multiple miRNA binding sites [125-126]. Simply due to their sequence diversity and the fact that they are predicted to target the majority of mRNAs in mammals, this regulatory pathway is of great importance. In fact, through a myriad of comparative expression analyses and gain- and loss-of function experiments, miRNAs have been shown to be critically involved in biological development, cell differentiation, apoptosis, cell-cycle control, stress response and disease pathogenesis [120, 127-129].

Given the roles of miRNAs in a wide variety of cellular processes, it would seem likely that these non-coding RNAs could play critical roles in metabolic

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Figure 9. General mechanism of translational regulation by microRNA. MicroRNAs are targeted to the 3â&#x20AC;&#x2122; UTR of specific mRNA transcripts. Depending on either perfect or imperfect base-pairing, microRNA:mRNA duplexes may targeted to degradation or temporary storage, respectively.

rate depression in anoxic turtles. For example, the activity of signalling networks, such as those mentioned in this chapter (JNK, ERK, p38 and PI3-K/Akt) can be susceptible to changes in protein abundance. The ability of miRNAs to influence protein amount, could yield significant control of the regulation of these signalling networks, effectively changing the signalling landscape and reprioritizing ATP metabolism during periods of stress [130-131].

Furthermore, a recent study examining the effect of protein synthesis for several thousand proteins showed that changes in a single miRNA can directly decrease the production of hundreds of proteins through a combination of mRNA storage and degradation [132]. This suggests that even a moderate change in miRNA expression may yield significant control over many metabolic processes known to be reduced in the anoxic turtle. In addition, widespread regulation by miRNA could also result in the reduction of translational rate (0-8% after 12 hours anoxia when compared to control values) as seen in hypometabolic turtles. As there is no change in the availability of translatable RNA, perhaps miRNA may establish a state of translational repression through mRNA storage in cytoplasmic storage granules (such as p-bodies or stress granules), rather than mRNA degradation [6, 107, 133-137]. Additionally, the extent to which mRNA-miRNA pairing

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occurs may allow the expression of key genes necessary for survival. For example, mRNA-miRNA interactions may allow for the translation of HSPs necessary for protein folding, while inhibiting expression of proteins involved in cellular proliferation. What remains to be discovered is the role of miRNA-mediated repression in regulating the global translational process facilitated through signaling pathways, such as the previously described PI3-K/Akt. Recent studies have documented changes miRNA expression patterns in two systems of natural hypometabolism: ground squirrel hibernation and freeze tolerance in wood frogs [138-139]. For example, studies examining the expression of miRNA during freeze tolerance in wood frogs found differential expression of miR-16-1 and miR-21 in liver and muscle tissues, two key microRNAs that play roles in arresting the cell cycle and inhibiting apoptosis, respectively. As it is of critical importance to reduce these ATPcostly processes during hypometabolism, miRNAs may act to aid in the reprioritization of ATP metabolism. Although miRNA research has not yet been carried out in turtles, existing research from other hypometabolic systems provides an indication that microRNAs may play a role in achieving a hypometabolic state among stress-tolerant turtles.

7.2. Prospects in longevity research Apart from the ability to escape environmental hardship by entering a hypometabolic state, turtles are also known for their extraordinary longevity.

Many turtle species survive longer than 100 years while displaying no known ageing-related diseases, such as the neurodegradation seen in Alzheimer’s patients. Important to longevity is the ability to inhibit or repress senescent phenotypes. A recent review from Krivoruchko and Storey, stated that turtles provide an intriguing model of negligible senescence displaying the following criteria; (1) mortality does not increase with age and (2) reproductive rates do not change with age [93]. As an example, one key study demonstrated that female painted turtles (C. picta marginata) (age 30-61 years) are able to lay more eggs and have more consistent annual reproduction rates, when compared to the average younger female turtle (age 9-19 years) [140]. Comparative studies carried out on Blanding’s turtles (E. blandingii) and the painted turtle, state that the Blanding’s turtle displayed very few of these characteristics. To this end, the Blanding’s turtle exhibited a reduction in offspring quality (egg and hatchling size) and survivorship with age, whereas painted turtles did not [140]. Therefore, only some turtles, such as C. picta marginata, appear to meet the criteria necessary for “negligible senescence”.

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The implications and causes of the ageing process are complex and seeking a single solution to remedy or decelerate the process will certainly end in failure. However, further understanding of individual processes that contribute to the overall ageing effect will enhance our knowledge and perhaps lead to treatments for age related diseases. Such a process includes the modification of ion channels in the presence of ROS leading to functional and structural changes to the channels affecting the ability to achieve ion homeostasis. Unfortunately, despite evidence correlating oxidative modification of ion channel activity and age-related neurodegeneration in humans, there are many conflicting results and a lack of comprehensive studies in the literature. Therefore, there is a clear need of further research examining the possible involvement of ion channels and of their modulation

by ROS during the ageing process, leading to a more systematic view of ageing and the role of ion channel modifications. Using the turtle as a model species, future studies can examine how these animals are able to maintain ion channel integrity despite bouts of potentially high levels of oxidative stress.

Apart from the deleterious effects of free radicals on the ageing process and the antioxidant defense system utilized by turtles, is the unique maintenance mechanisms of the turtle genome by telomerase. Telomerase is a ribonucleoprotein complex responsible for the maintenance of chromosome ends (telomeres) and for the repair of DNA strand breakage. In 1991, studies

by Blackburn showed that each round of cell division lead to a shortening of telomeres, effectively measuring cellular life span [141]. Once a critical threshold is reached, cell division ceases and the Rb/p16INK4a pathway is activated, leading to cellular senescence [142]. Additionally, in 2005 Girondot and Garcia reported that the turtle possesses very large telomeres and a somatic expression of the telomerase activity, presumably to counteract the mechanism of telomere shortening during cell division [142]. However, this study did not report the possibility of the antioxidant mechanisms, as utilized by the turtle to survive oxygen reperfusion, aiding in the preservation of telomere integrity; low oxidative stress has been shown to help maintain telomerase activity [143]. In 2004, Kurz and colleagues showed that in human umbilical vein endothelial cells (HUVEC) (used because of their high susceptibility to oxidative stress) persistent oxidative stress accelerates telomere shortening and the loss of telomerase activity [143]. Also, glutathione may play a pivotal role in the preservation of telomere integrity [144]. In light of recent research on telomere regulation during oxidative stress, it would be of interest to examine telomerase activity and telomere shortening in the turtle and its regulation during anoxia and metabolic rate depression.

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8. Concluding remarks Although much is known about the physiological responses of turtles during hypometabolism, studies evaluating the regulation of anoxia-induced gene expression during the transitions to and from this state are beginning to explore new and fascinating areas of molecular research. These findings have begun to develop a general, but refined, view of the important molecular pathways contributing to stress-survival. However, new studies utilizing broadly focused genomic and proteomic microarray screening, are identifying many new targets for future study. Many of these identified targets are intriguing to the comparative molecular biologist, and this technology provides the means to access the global expression of nearly all genes and proteins that contribute to anoxia-tolerance in turtles. Although there are many areas of study left to be explored, research in the hypometabolic responses of the turtle will always be continuing since new technologies allow further analysis of cell function, and new paradigms in gene regulation and regulatory molecules (such as microRNAs) are continuing to be discovered. Building upon the discoveries of past research, future studies can be carried out in a variety of different areas still left unexplored, creating a more complete understanding of the anoxic survival mechanisms utilized by the turtle.

Acknowledgments Thank you to JM Storey for critical commentary and editorial review of the manuscript. Research in the Storey lab is supported by a discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to KBS and the Canada Research Chairs program. KKB held a NSERC CGSD scholarship and AGG held a NSERC USRA scholarship.

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Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates, 2011: 95-115 ISBN: 978-81-308-0471-2 Editors: Anna Nowakowska and MichaĹ&#x201A; Caputa

5. Maintaining metabolic balance in mammalian hibernation and daily torpor James F. Staples Department of Biology, University of Western Ontario, London, ON N6A5B8, Canada

Abstract. Hibernation and daily torpor are hypometabolic states that involve substantial decreases in body temperature (Tb) and whole-animal metabolic rate. Metabolic suppression is almost certainly an active, regulated process, as it precedes any drop in Tb, and is spontaneously reversible, even at low Tb. While reduced thermogenesis and passive thermal effects contribute to decreases in metabolic rate, evidence suggests tissue-specific metabolic suppression below basal levels. Within these hypometabolic states, the balance between tissue ATP production and consumption is largely maintained, suggesting coordinated downregulation. ATP consumption, by such processes as ion pumping and protein turnover, is downregulated in a tissue-specific and hierarchical manner by combinations of active regulation (e.g. posttranslational modification) and passive thermal effects as Tb falls.

Glycolysis and carbohydrate oxidation are decreased by post-translational modification of rate controlling enzymes.

Mitochondrial metabolic suppression is best described in liver, where oxidative phosphorylation may decrease by 70% as animals make the transition from the euthermic to torpid states.

Correspondence/Reprint request: Dr. James Staples, Department of Biology, University of Western Ontario London, ON N6A5B8, Canada. E-mail: jfstaple@uwo.ca

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Mitochondrial substrate oxidation, especially through electron transport chain complex II, is reversibly suppressed in torpid animals. This oxidative suppression may involve allosteric or covalent inhibition of succinate dehydrogenase and other electron transport chain complexes. In hibernation there is no apparent suppression of mitochondrial oxidative metabolism in skeletal muscle, but little reliable data is available for daily torpor or for other tissue types. The mitochondrial membranes of hibernators are transiently remodelled during the transition from euthermia to torpor, but the functional significance of these changes remains to be discovered.

Introduction Hibernation and daily torpor are found in at least eight mammalian orders [1]. By lowering whole-animal metabolic rate (usually measured as mass-specific oxygen consumption rate; V· O2), and allowing core body temperature (Tb) to fall, animals can conserve up to 88% of the energy that would be required to remain euthermic at a typical endothermic Tb [2].

From late autumn through early spring hibernators experience many torpor bouts, each consisting of four phases: 1) entrance, where Tb and V· O2 decrease rapidly over a few hours, 2) torpor, where Tb and V· O2 remain low and fairly constant for at least several days, 3) arousal, where Tb and V· O2 spontaneously increase rapidly, and 4) interbout euthermia, where Tb and V·O2 are at high and fairly stable values for about a day. (Figs. 1, 2). Obligate hibernators, including the well-studied ground squirrels, hibernate every year, regardless of environmental conditions. In Syrian hamsters (Mesocricetus auratus), however, hibernation is facultative, and is potentiated by short photoperiod, cold ambient temperatures and food restriction.

Bouts of daily torpor last less than 24 hours, and Tb and V· O2 remain above 10oC and 20% of resting levels, respectively [1]. However the degree of V· O2 suppression in both hibernation and daily torpor depends on many factors and scales allometrically with body mass (see [1] for a thorough review). In some daily heterotherms, such as the dwarf Siberian hamster, Phodopus sungorus, daily torpor occurs spontaneously after acclimation to cool temperatures and short photoperiod, even when food is provided ad libitum. However, in other mammals, such as the little brown bat, Myotis lucifugus, the pattern, if not the expression, of daily torpor is affected by energy availability [3]. In contrast, mice (Mus musculus), normally remain euthermic when fed ad libitum, but when fasted, enter reversible daily torpor [4].

Figure 1. Patterns of hibernation and daily torpor. Fig 1A shows core body temperature during several torpor bouts for the hibernator Ictidomys tridecemlineatus. The different phases of one bout are labelled: 1) entrance, 2) torpor, 3) arousal, 4) interbout euthermia. Fig 1B shows body temperature of two P. sungorus. One animal displayed spontaneous daily torpor (dashed line) while the other (solid line) remained euthermic (from [5]). The torpid animal was sampled during the second torpor bout, at the point indicated by arrow. Fig. 1C shows body temperature for an individual Mus musculus during fastinginduced daily torpor. Prior to fasting (first arrow) food was available ad libitum and the mouse did not display torpor. Upon refeeding (second arrow) the animal returned to euthermia and did not display torpor. Black bars above the time axis indicate the scotophase (from [4]).

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Control of the initiation and termination of torpor bouts in hibernators and daily heterotherms has been an area of intense research effort at least since the 1960â&#x20AC;&#x2122;s (e.g. [6]). Moreover the extreme metabolic cycles observed among torpor bout stages are superimposed on seasonal cycles that regulate, among other things, feeding and fasting [7]. Extensive coverage of this topic is beyond the scope of this review, but it is important to note the importance of endogenous circannual rhythms in determining when the hibernation season begins in obligate hibernators. For example I. tridecemlineatus, enter torpor in the autumn, even when housed under constant summer conditions (20oC, 14L:10D; [8]). It is clear that certain protein concentrations change in tissue and blood plasma in the different phases of hibernation torpor bouts (e.g. [9]), but the functional significance of these oscillations remains unclear. It has been hypothesized that any of several brain neuromodulators

Figure 2. Metabolic rate and body temperature in hibernation and daily torpor.

Fig. 2A shows the entrance stage of a torpor bout in I. tridecemlineatus. Fig 2B shows daily torpor in P. sungorus over approximately 2 days during which the animal entered two bouts of torpor. The black bars above the time axis indicate the scotophase.

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may play a role in controlling torpor bouts in hibernators. These compounds include adenosine, glutamate, histamine, µ opioids and thyrotropin releasing hormone (reviewed in [10]). For daily torpor there is little data available regarding the regulation of torpor bout initiation, except to note that in mice that undergo fasting-induced torpor, injected AMP does not initiate torpor [11].

In both hibernation and daily torpor (Fig. 2) the decrease in V· O2 during entrance, and increase during arousal precede any changes in Tb. During these transitions, changes in the thermoregulatory set-point of the pre-optic anterior hypothalamus [12,13] cause changes in shivering and non-shivering thermogenesis which contribute significantly to changes in V· O2. As Tb falls during entrance, V· O2 would decrease further by passive thermal effects on enzyme-catalyzed reactions. So is there any reason to predict an active, regulated metabolic suppression of tissue metabolic in hibernation and daily torpor beyond changes in thermogenic metabolism and passive thermal effects? One response to this question comes from the edible dormouse, Glis glis, which, when held at thermoneutral temperatures (28.6oC), will enter daily torpor. Under these conditions, entrance is associated with a considerable decrease in V· O2 before Tb changes, suggesting an active suppression on non-thermogenic (i.e. basal) metabolism [14]. The available data suggest that regulated metabolic suppression accounts for 40-70% of the total decrease of V· O2 in hibernators and 14-30% in daily heterotherms [14,15], with passive thermal effects accounting for the remainder of the observed decrease in V· O2.

Metabolic balance is maintained and regulated during hibernation hypometabolism Tissue contents of high-energy phosphates depend largely on a balance between fluxes of pathways that produce and consume ATP. Given the reduced whole-animal V· O2 seen in hibernation and daily torpor it is reasonable to assume that mitochondrial oxidative phosphorylation is suppressed to a similar extent. Without corresponding reductions in ATP consuming pathways one would predict a steady decline in tissue high-energy phosphates.

Liver [ATP] and [ADP] do not change among the different stages of torpor bouts in the hibernating golden-mantled ground squirrel, Callospermophilus lateralis [16]. In another ground squirrel, Ictidomys tridecemlineatus, liver [ATP] has actually been reported to increase during hibernation [17]. Using 1H-NMR on living animals, Henry et al. [18] found that brain creatine phosphate (CrP) is 33% higher in I. tridecemlineatus brain during torpor and interbout euthermia compared with fall and spring

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euthermic animals. Even during arousal, where whole-animal V路 O2 and tissue ATP-turnover increase massively, 31P-NMR shows that liver [ATP] does not change [19].

Contrasting these data, however, are reports that show [ATP] and [CrP] decrease by about 40% during hibernation in the C. lateralis skeletal muscle [20] and the brain of Zapus hudsonius (jumping mouse; [21]). These results suggest tissue-specific differences in the degree of metabolic balance maintained during hibernation even in the same species. Regardless, it appears that tissue metabolism remains tightly regulated. For example, the reported decreases in [ATP] in hibernator tissues are paralleled by decreases in total adenylates and are not accompanied by accumulations of ADP or AMP [20,21]. As a result ATP:AMP, important in regulating enzymes such as AMP kinase, does not change during hibernation, suggesting the maintenance of tight regulation of tissue metabolism. However, in Jaculus orientalis skeletal muscle ATP:AMP has been reported to decrease in hibernation [22].

For the most part, data suggest that metabolic balance and regulation are maintained throughout hibernation bouts. Unfortunately there are relatively few studies available, and these have used diverse models, tissues and measurement techniques. To my knowledge similar data for daily torpor are lacking. The apparent maintenance of metabolic balance implies that any suppression in ATP production must be matched by a corresponding suppression in ATP consumption.

Reducing ATP consumption Rolf and Brand [23] estimated that, in mammals, oxygen consumption coupled to ATP synthesis is accounted for by the following processes: Na+/K+ ATPase (up to 30% of total O2 consumption), protein synthesis (up to 28%), Ca2+ ATPase (8%), actinomyosin (10%), gluconeogenesis (10%) and ureagenesis (3%). Reducing the demand for these processes in hibernation and daily torpor would help to maintain metabolic balance. These pathways actually consume ATP, but other metabolically expensive processes not coupled to ATP synthesis could potentially be downregulated to conserve energy during hibernation and daily torpor.

Ion pumping The maximal activity of Na+/K+ ATPase decreases in hibernation in several ground squirrel tissues [20,24], although this pattern is not seen in the

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kidney of another hibernator, J. orientalis [25]. Such decreases may be due to reduced amounts of Na+/K+ ATPase protein [24], but the activity and kinetics of Na+/K+ ATPase can also be acutely regulated by phosphorylation [20].

Na+/K+ ATPase activity can also be altered by changes in the membrane phospholipid environment in which the enzyme works [26,27], so that, although in vitro Na+/K+ ATPase Vmax increases in hibernating Urocitellus richardsonii brain [26], its function in vivo may actually be downregulated.

Cellular Ca2+ appears to be regulated in a tissue-specific manner in hibernators. Rates of Ca2+ accumulation are significantly lower in synaptosomes isolated from the brains of hibernating ground squirrels compared with control cold-acclimated euthermic animals, probably due to down regulation of voltage-gated Ca2+ channels [28]. Such alterations would presumably reduce ATP demand for Ca2+ pumping, and could contribute to the apparent hypoxia tolerance of hibernator brain [29]. Neuromodulators such as NMDA evoke much slower Ca2+ uptake in hippocampal cells from hibernating Mesocricetus auratus compared with active conspecifics [30]. In cardiac tissue, however, energy devoted to cellular Ca2+ homeostasis may be increased in hibernation.

In non-hibernating mammals, hypothermic death usually results from heart failure due to the inability to remove myocardial cytosolic Ca2+. In ground squirrels, however, expression of SERCA, the enzyme primarily responsible for Ca2+ reuptake, increases dramatically in the hibernation season. At the same time expression of an important SERCA inhibitor, phospholamban, declines (reviewed in [31]). In another vertebrate model of hypometabolism, the hypoxia-tolerant turtle Trachemys scripta, SERCA activity and kinetics are thought to be acutely regulated by post-translational modification during the transition from normoxia to hypoxia [32]. To my knowledge, however, this aspect of SERCA regulation has not been explored in mammalian hibernators or daily heterotherms.

In other excitable tissues, such as brain, reducing the frequency of action potentials could significantly decrease the cost of ion homeostasis. In hibernators, while some brain regions may actually increase relative rates of metabolism during the torpid stage [10,33], electroencephalographic activity generally decreases. This â&#x20AC;&#x153;spike arrestâ&#x20AC;? is not readily explained by changes in inhibitory neuromodulators. Indeed extracellular concentrations of GABA actually decrease during hibernation in Spermophilus parryii [34]. In addition the sensitivity to adenosine decreases during hibernation in Mesocricetus auratus, suggesting a decrease in available adenosine receptors [35].

Therefore the observed decreased brain electrochemical activity in hibernation is thought to be caused primarily by passive effects of low Tb [36].

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Protein turnover and gene transcription Protein synthesis accounts for up to 28% [23] of mammalian cellular O2 consumption, so ATP turnover could be reduced considerably in hibernation and daily torpor if this cost could be reduced. Several enzymes involved in protein synthesis are present in I. tridecemlineatus liver at higher levels during entrance than in summer-active animals [37]. Despite this result, protein synthesis is reversibly downregulated during arousal and the torpor phase of ground squirrel hibernation in brain [38] and liver [39]. Much of this downregulation is probably due to low Tb [39], but phosphorylationdependent inhibition of translation may also be important [40], along with an inhibition of elongation [38]. Translation in liver tissue is also reversibly suppressed during daily torpor in P. sungorus [41] as is proteolysis in hibernating ground squirrels, where low temperature likely plays a significant role [42,43].

Gene transcription is estimated to account for up to 10% of mammalian cellular ATP turnover [23]. Initiation of transcription is suppressed by half in hibernating C. lateralis, and elongation is very temperature-sensitive, so that very little transcription is likely to occur at typical hibernation or daily torpor Tb [44].

Mitochondrial proton conductance The mitochondrial electron transport chain (ETC) pumps protons from the mitochondrial matrix to the intermembrane space, establishing the proton motive force (ΔP) across the inner mitochondrial membrane (IMM). When ATP turnover and ADP supply rate are high, ΔP powers ATP synthesis through the F1FOATP synthase, stimulating flux through the ETC and, subsequently, substrate oxidation (Fig. 3). Under conditions of low ATP turnover ΔP is high, ETC complexes are highly reduced, and electrons from I and III may react with O2 to form superoxide, which may be metabolized to other forms of reactive oxygen species (ROS). Under these conditions a “leak” of protons across the IMM is thought to decrease the reduction state of complexes I and III and reduce ROS production. This proton conductance would result in a persistent, albeit low O2 consumption under conditions of low ATP turnover.

Although a mechanism for proton leak remains unclear (see [46-49]) it may account for 22% of mammalian cellular respiration rates [50]. In isolated liver mitochondria Barger et al. [51] reported no change in IMM proton permeability in hibernating U. undulatus whereas Gerson et al. [52] did find a significant decrease in proton leak in hibernating I. tridecemlineatus, though

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Figure 3. A diagrammatic representation of the electron transport chain (ETC).

NADH and succinate donate electrons at complexes I and II, respectively. As electrons are transferred down the ETC (denoted by thin black arrows), protons are pumped from the matrix of the mitochondria (from complexes I, III and IV) to the intermembrane space (IMS) establishing a proton motive force. This proton motive forces is used by F1FO ATP synthase (Complex V) to phosphorylate ADP to ATP. Figure from [45].

this result depends on dietary polyunsaturated fats. In U. undulatus skeletal muscle UCP3 expression increases during hibernation, but proton leak kinetics do not change [53]. During daily torpor in P. sungorus proton leak actually increases (Fig. 4), perhaps as a mechanism to reduce reactive oxygen species production [5]. Despite this increase in proton leak state 3 mitochondrial respiration in torpid P. sungorus is actually lower, probably due to a decreased substrate oxidation capacity (Fig. 4; [5]).

Although the permeability of the IMM to protons may not be actively altered in hypometabolic animals, other metabolic changes may affect it indirectly. Proton conductance increases exponentially with ΔP (the “nonohmic” pattern; [54]), so decreasing ΔP would substantially decrease the rate at which protons leak across the IMM. Indeed liver mitochondria from hibernating U. undulatus have lower maximal ΔP than active controls under non-phosphorylating (e.g. no ATP turnover) conditions [51]. A reduction in

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Figure 4. Proton leak increases in daily torpor. Kinetics of mitochondrial proton leak measured at (a) 37oC and (b) 15oC for liver mitochondria from P. sungorus sampled in normothermia and torpor. Values are mean Âą SD. Sample sizes are indicated in parentheses beside each curve.

Î&#x201D;P can be achieved, at least in part, by decreasing rates of substrate oxidation, illustrating how a decrease in energy supply can be linked to decrease in energy demand.

Reducing ATP supply Important rate-controlling glycolytic enzymes, such as phosphofructokinase and pyruvate kinase, consistently show post-translational modification and decreased activities during hibernation in ground squirrels (reviewed in [55]).

These changes are accompanied by changes in the enzymes likely responsible for phosphorylation â&#x20AC;&#x201C; protein kinase C and A [55]. Pyruvate dehydrogenase activity is downregulated by covalent modification in hibernating ground squirrel heart and kidney [56] and P. sungorus heart, BAT and liver during

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daily torpor [57]. Although such alterations probably serve to switch substrate preference away from carbohydrate and toward lipid [57,58], they may also reflect reduced oxidative substrate supply to mitochondria.

It appears that mitochondrial oxidative capacity is suppressed in hibernation and daily torpor, but in a tissue-specific pattern. There are conflicting reports about whether brown adipose tissue (BAT) mitochondrial oxidative capacity is reversibly suppressed between the interbout euthermic and torpor stages of hibernation [59,60]. However, in the absence of adrenergic stimulation during the torpor phase, BAT activity would be minimal and any further active suppression of BAT mitochondrial metabolism would likely contribute little to whole-animal energy savings. Skeletal muscle mitochondrial respiration has been reported not to change when compared between hibernating and summer-active I. tridecemlineatus [61], and hibernating and winter-active (i.e. had not displayed torpor bouts) U. undulatus [51]. However, we recently found a 33% reduction in skeletal muscle mitochondrial metabolism when comparing torpid and IBE I. tridecemlineatus (Fig 5; J. Brown, D. Chung, K. Belgrave, J. Staples, unpublished). In contrast liver mitochondria isolated from hibernating ground squirrel display state 3 respiration that is reduced by up to 70% [45,51, 61-63], and we have found similar reductions in state 4 respiration [45]. This inhibition of liver mitochondrial metabolism is reversed during arousal [45,64].

Figure 5. Suppression of skeletal muscle mitochondrial metabolism in a hibernator.

Mitochondria were isolated from mixed hind-limb muscles of I. tridecemlineatus sampled either 3-4 days into a torpor bout, or during interbout euthermia. Respiration rates (state 3 filled bars, state 4 open bars) were measured at 37oC with 6mM succinate and 0.5ÂľM rotenone. Values are means Âą SEM. Asterisk indicates significant difference from interbout euthermia (t-test, P<0.05).

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This pattern of reversible inhibition of mitochondrial oxidative metabolism may offer clues to the mechanisms involved. Recently we measured aspects of oxidative metabolism in liver mitochondria isolated from I. tridecemlineatus in different stages of a torpor bout. In the short time between interbout euthermia and early entrance (Tb 30oC) succinate-fuelled state 3 respiration, measured in vitro at 37oC, decreases by approximately 60% [65]. However, there is no further suppression in late entrance (Tb 15oC) or torpor (Tb 5oC). By contrast, during arousal, the reversal of mitochondrial metabolic suppression is gradual. Early in arousal (Tb 15oC) state 3 respiration is very low, and not significantly different from torpor [45]. By the time Tb increases to 30oC late in arousal, respiration has increased 3-fold relative to torpor, but continues to increase into interbout euthermia (Fig. 6).

The rapid suppression of mitochondrial oxidative metabolism during entrance at relatively high Tb, and its slow reversal during arousal when Tb is lower suggests that mechanisms of metabolic suppression are temperaturedependent, i.e. at high temperatures they can be implemented rapidly, but at low temperatures reversal occurs slowly.

Figure 6. A representation of liver mitochondrial state 3 respiration rates (measured in vitro at 37oC with rotenone and 6 mM succinate) during different stages of a typical torpor bout in I. tridecemlineatus. When mitochondria are isolated from animals in torpor, respiration is low. During arousal respiration increases, but not until interbout euthermia, where Tb is ~ 37oC does it reach maximal values. In contrast respiration is rapidly and maximally suppressed in the early stages of torpor when Tb is still fairly high.

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Potential mechanisms of reversible suppression of mitochondrial metabolism Succinate oxidation is reduced in P. sungorus during spontaneous daily torpor [5]. In fasting-induced daily torpor, however, suppression of substrate oxidation depends on the substrate (i.e. glutamate vs. succinate) and differs among strains of Mus musculus [4]. Moreover suppression of the mitochondrial ADP phosphorylation system may contribute to energetic saving in fasting-induced daily torpor, but in at least one strain of Mus musculus the activity of the phosphorylation system is actually higher in torpid than euthermic animals [4].

Mitochondrial substrate oxidation is suppressed in hibernation, but the degree of suppression appears to depend on respiratory substrate, giving clues as to where inhibition may occur. Substrates that feed reducing equivalents to ETC complexes I (e.g. pyruvate, glutamate, β-hydroxybutyrate) and II (e.g. succinate) generally result in reduced state 3 respiration rates in hibernation [62,66-68]. Suppression is less evident with substrates that donate electrons directly to complex IV [66,68]. Daily torpor in P. sungorus produces similar results; significant suppression of state 3 respiration with glutamate and succinate, but not with decylubiquinone (which directly reduces complex III) or ascorbate and TMPD (directly reducing complex IV; [5]). These data could result from decreased activities of specific substrate transporters, substrate dehydrogenases (supplying fuel to the Krebs cycle), Krebs cycle enzymes (generating reducing equivalents for the ETC), or from a decrease in ETC capacity or function between complexes I and IV. Substrate transport is known to control isolated mitochondrial succinate oxidation but only at very low succinate concentrations [69]. Studies that have demonstrated respiratory suppression in hibernation typically use succinate concentrations of 5-6mM [51,63,68], close to the estimated intramitochondrial levels of 4mM [69], so that transport should not be limiting. Carboxylate transporters are known to exhibit considerable control over rat liver mitochondrial state 3 respiration [70]. To my knowledge, however, there is no information available about changes in substrate transporters in hibernation or daily torpor, except to note that protein levels of carnitine palmitoyl transferase-1β (important in transporting long-chain fatty acids into the mitochondria) are upregulated in BAT, but not skeletal muscle in hibernating Myotis lucifugus [71].

Krebs cycle activity is thought to be primarily controlled by substrate availability, therefore decreased activities of substrate dehydrogenases in hibernation and daily torpor could limit carbon fuel supply to the Krebs cycle. Pyruvate dehydrogenase activity is downregulated by covalent modification in hibernating ground squirrel heart and kidney [56] and

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P. sungorus heart, BAT, and liver during daily torpor [57]. Recently it was reported that the activity, kinetics and phosphorylation state of glutamate dehydrogenase, a mitochondrial matrix enzyme, differ between euthermic and torpid grounds squirrels [72]. However, in a variety of P. sungorus tissues, including liver, maximal glutamate dehydrogenase activity does not differ between euthermia and torpor [57], perhaps indicating a difference in the regulation of mitochondrial metabolism between hibernation and daily torpor. The activity and amount of liver β-hydroxybutyrate dehydrogenase increases during hibernation in J. orientalis [73], but the significance of this is questionable as β-hydroxybutyrate oxidation rates were not measured.

Indeed in U. undulatus [62,66-68] and I. tridecemlineatus [61] oxidation of β-hydroxybutyrate by liver mitochondria is decreased substantially in hibernation relative to the summer active state. The activity of β-hydroxyacylCoA dehydrogenase (HOAD; an important fatty acid β-oxidation enzyme supplying acetyl-coA to the Krebs cycle) increases in hibernation in skeletal muscle [74,75] but to our knowledge there are no data available for liver.

Surprisingly there is very little data available on the activity and regulation of Krebs cycle enzymes in hibernation or daily torpor. The activity of α-ketoglutarate dehydrogenase appears to be decreased in liver mitochondria isolated from hibernating Mesocricetus auratus [64]. Liver citrate synthase activity decreases during hibernation in I. tridecemlineatus [76] and the bat Murina leucogaster [77], though no difference is seen 1 month after final arousal in another bat, Eptesicus fuscus [78]. Citrate synthase activity is reported to increase during hibernation in pectoralis muscle of bats [74,79] and leg muscles of C. lateralis and Mesocricetus auratus [75,80]. The significance of these data to hibernation metabolic suppression is unclear, however, as respiration of muscle mitochondria isolated from hibernating ground squirrels are not different from active controls [51,68]. While it is known that citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are activated by Ca2+, little is known about mitochondrial Ca2+ content in hibernation except to note that liver mitochondrial Ca2+ uptake is decreased in hibernation [81]. These enzymes are also regulated by redox state, but again to our knowledge there is no functional data available about how mitochondrial NAD+/NADH changes with hibernation status.

In C. lateralis, nad2 (mitochondrial-encoded ETC complex I subunit) mRNA is upregulated in heart, liver and skeletal muscle during hibernation [82]. The significance of altered gene transcription to mitochondrial function is far from clear, however, and the reported pattern may be a

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response to low temperatures rather than hibernation per se [82] or an anticipatory preparation for the arousal phase. The activity of complex I is also regulated by cyclic AMP-dependent phosphorylation [83], so we are currently characterizing the phosphorylation status of ETC complex subunits in liver mitochondria purified from I. tridecemlineatus in torpor and interbout euthermia, and relating this status to respiration rates. In contrast, there is no difference in complex I Vmax between liver mitochondria isolated from normothermic and torpid P. sungorus, even though oxidation of complex I-linked substrates is significantly reduced [5].

Again this may suggests potential differences in the regulation of mitochondrial metabolism between hibernation and daily torpor.

The Vmax of succinate dehydrogenase (SDH, ETC complex II) decreases by 72% in liver mitochondria isolated from torpid I. tridecemlineatus relative to interbout euthermia [45]. Moreover succinate-fuelled state 3 and state 4 respiration in various stages of a torpor bout correlate strongly with SDH activity [45,65], suggesting a strong role for this substrate oxidation step in controlling mitochondrial metabolism in hibernation. Interestingly the regulation of SDH may differ depending on the stage of a torpor bout.

Incubation with succinate plus isocitrate relieves some of this respiratory suppression in torpor, suggesting an accumulation of oxaloacetate during torpor is partially responsible for the observed SDH inhibition [45]. Indeed SDH activity and state 3 respiration correlate negatively with liver oxaloacetate content during arousal in I. tridecemlineatus [45]. However, during entrance, isocitrate incubation has little effect on SDH activity or mitochondrial respiration [65], suggesting that other mechanisms may be more important when entering a torpor bout. Recently it has been reported that SDH activity can be reversibly regulated in an acute manner by phosphorylation of a flavoprotein subunit [84], and we are investigating this possibility in liver mitochondria from I. tridecemlineatus.

ETC flux through complexes I and II could also be affected by coenzyme Q, which transfers electrons to complex III within the IMM. In fact one study suggests that the flux from coenzyme Q to cytochrome bc1 limits respiration in liver mitochondria from hibernating U. undulatus [85]. However the concentration of coenzyme Q in liver mitochondria does not differ between hibernating and non-hibernating I. tridecemlineatus [62]. In I. tridecemlineatus, liver mitochondrial cytochrome b levels decrease by 25% in hibernation, but there is no change in cytochrome c content [62]. Both spectroscopic [62] and Vmax measurements [52] suggest that the total amount of liver complex IV does not change with hibernation in I. tridecemlineatus. Despite these findings respiration with ascorbate and TMPD is significantly inhibited in torpor and arousal [61], suggesting acute inhibition of complex IV.

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Membrane composition can affect mitochondrial function by effects on, among other things, fluidity and membraneâ&#x20AC;&#x201C;bound enzyme activities [86]. In mammalian hibernators, Tb changes by over 30oC within a matter of hours (Fig. 1), but even during this short time frame hibernator membranes are remodelled. For example the phosphatidylethanolamine (PE) content of brain synaptosomes decreases following induced arousal in arctic ground squirrels [87]. While these changes may be important for maintaining membrane fluidity as Tb changes [88], some phospholipids have profound impacts on mitochondrial function. For example cardiolipin is known to affect the activity of several inner mitochondrial membrane proteins including ETC complexes I and III and the adenine nucleotide transporter (reviewed in [88]).

To investigate potential effects of membrane composition on mitochondrial function in hibernation we characterized the phospholipid content of liver mitochondria purified from the liver of I. tridecemlineatus in different stages of a torpor bout. During arousal the relative content of phosphatidylcholine (PC) increases while PE decreases during arousal.

During arousal monounsaturated fatty acyl constituents of phospholipids do not change, but polyunsaturates increase [90]. While state 3 respiration

Figure 7. The relationship between phospholipid content of palmitoleic acid (16:1) and succinate-fuelled state 3 respiration in liver mitochondria purified (see [45] for methods) from I. tridecemlineatus in different stages of hibernation. In the hibernation season animals were sampled in torpor (Tb steady ~5oC), early arousal (Tb=15oC and rising), late arousal (Tb=30oC and rising) or interbout euthermia (Tb steady ~37oC).

Some animals were also sampled in the summer when they would not hibernate.

There is a significant positive relationship (Pearson product moment correlation).

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increases throughout arousal (Fig. 6) the only phospholipid characteristic that correlate significantly with state 3 was the relative content of palmitoleic acid (16:1; Fig. 7).

The pattern of membrane phospholipid remodelling during entrance into a torpor bout differs fundamentally from arousal [65]. PC content decreases by approximately 40% between interbout euthermia and early entrance (Tb=30oC), but by late entrance (Tb=15oC) recovers to levels that do not differ from interbout euthermia or torpor. This transient phospholipid remodelling is reflected by increases and decreases in major saturates and polyunsaturates, respectively, during early entrance, followed by recovery in late entrance and torpor to levels no different from interbout euthermia. Unlike arousal, no phospholipid characteristics correlate significantly with succinate-fuelled respiration, despite the fact that state 3 respiration falls by approximately 60% between interbout euthermia and early entrance [65].

Future directions In hibernators the suppression of mitochondrial respiration occurs quickly during entrance into a torpor bout, when Tb is relatively high. It is reversed during arousal, but only gradually as Tb climbs. Together these observations suggest acute, temperature-dependent, enzyme-catalyzed alterations of mitochondrial pathways mediate this extreme metabolic plasticity. It would be informative, therefore, to examine how the phosphorylation and acetylation states of Krebs cycle and ETC enzymes change throughout a torpor bout. We are currently characterizing the phosphorylation status of ETC subunits in various tissues sampled from I. tridecemlineatus in different stages of a torpor bout using 2D native blue gel electrophoresis to separate ETC complexes and subunits. We will then use specific antibodies to characterize the phosphorylation status of these proteins.

While liver mitochondria have been extensively studied in hibernators, and may offer insights into potential mechanisms of metabolic suppression, the response of mitochondria from other tissues is largely unknown. It would be informative, therefore, to measure mitochondrial responses to hibernation and daily torpor in tissues that contribute significantly to whole-organism metabolic rate. To that end we are currently using saponin-permeabilized brain slices [91] to compare mitochondrial function between I. tridecemlineatus in interbout euthermia and torpor. Similar studies on other tissues, including heart and kidney, will improve our understanding of the role of mitochondrial suppression to overall energy saving is hibernation.

Similar experiments on daily heterotherms would be useful for identifying potential similarities and difference between the two phenotypes.

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It is interesting to note that the metabolic suppression observed in liver mitochondria of hibernators [61] and daily heterotherms [5] depends on the temperature at which these in vitro measurements. For example, when measured at 37oC, state 3 respiration is 70% lower in mitochondria from torpid animals compare to summer-active animals. When measured at 25oC, respiration rates are lower in both groups, but the difference between torpor and summer-active is diminished. At 10oC there is no difference between the torpid and active states. We have interpreted this to mean the active suppression of mitochondrial metabolism is most important in the early stages of entrance, and passive thermal effects become more important to reducing metabolism at low the low Tb throughout most of the torpor bout.

These results also imply that active suppression of metabolic pathways may be more important to energy savings in hypometabolic animals that do not experience the extreme drops in Tb observed in small hibernators. For example it was recently reported that, in hibernating black bears (Ursus americanus), V路 O2 may decrease by 75%, but Tb rarely falls as low as 30oC [92]. It would, therefore, be of great interest to study how tissue oxidative metabolism is regulated in hibernating bears.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates, 2011: 117-145 ISBN: 978-81-308-0471-2 Editors: Anna Nowakowska and Michał Caputa

6. Hypometabolism as a strategy of survival in asphyxiated newborn mammals Justyna Rogalska and Michał Caputa Department of Animal Physiology, Institute of General and Molecular Biology, N. Copernicus University, ul. Gagarina 9, 87-100 Toruń, Poland

Abstract. Newborn mammals have to withstand the stress of being born, during a variable period of limited oxygen supply. We will discuss the mechanisms that allow asphyxiated newborn mammals to survive, with a particular emphasis on metabolic rate depression as the key survival strategy for enduring interruptions of oxygen supply.

Neonatal hypoxic hypometabolism is accompanied by anapyrexia (reduced level of body temperature regulation). These two processes reinforce each other in the protection against neonatal asphyxia. Another, complementary physiological adaptation, which enables newborns to cope with asphyxia and to autoresuscitate when oxygen is made available, seems to be their unique gasping ability. A long-term decrease in body temperature of newborn rodents, combined with their gasping ability, makes them extremely tolerant to asphyxia. Elucidation of the mechanisms responsible for perinatal hypoxic tolerance of newborn animals might shed some light on treatment of asphyxiated humans.

Correspondence/Reprint request: Dr. Justyna Rogalska, Department of Animal Physiology, Institute of General and Molecular Biology, N. Copernicus University, ul. Gagarina 9, 87-100 Toruń, Poland E-mail: rogal@umk.pl

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1. Introduction Newborn mammals have to cope with perinatal hypoxic stress when they transfer from maternal delivery of oxygen and nutrients to independent breathing. During this event newborns face the potential danger of becoming hypoxic to the point of causing irreversible damage to the central nervous system. However, it is well known that for the first few days after birth, the brain of the mammalian neonate is much more resistant to hypoxia than that of the adult [1,2,3,4,5]. Rodents can afford not only multiple pregnancy (in case of rats up to 30 healthy newborns in a litter) [6] but also breeding at altitudes as high as above 4300 m [7] and even give birth in burrows where the concentration of oxygen may be as low as 10 % [8]. Cortical and hippocampal neurons of rats are hypoxia-tolerant during the embryonic and neonatal periods [9,10]. Newborn rats can survive in a pure nitrogen atmosphere as much as 30 times longer than adults of the same species [1].

Since most newborns survive parturitional asphyxia without sequelae there must be inborn strategies for tolerating the impaired gas exchange.

Metabolic rate depression is a widespread response to anoxia. It is a key component of anoxia survival especially in anoxia-tolerant organisms. Since this mechanism of defence against asphyxia is similar among reptiles, birds and mammals [11], we can state that the lower rate of O2 consumption can also underlie the hypoxia tolerance of the neonate brain.

Under hypoxic conditions metabolic rate is lowered to a level that can be supported by the ATP output from fermentative pathways alone. For example, turtles submerged under water suppress their anoxic metabolic rate to 10 to 20% of their resting metabolic rate when breathing air at the same temperature and in marine mollusks anoxic metabolic rate is reduced to only 2 to 10% of the aerobic value [11].

More than 50 years ago, Cross et al. [12] noticed that newborn infants decrease their oxygen consumption (VO2) when breathing 15% O2. Hypoxic hypometabolism is also characterized by a substantial drop in body temperature [12], and an attenuation of both hyperventilation and cardiac activity, which are oxygen consuming [13,14]. Studies on newborn kittens and rats [15,16] indicated that hypoxic hypometabolism occurred even at moderate levels of hypoxia. In addition, they demonstrated that ambient temperature was a factor determining magnitude of the hypometabolic response to hypoxia. These studies confirmed that hypometabolism and the drop in body temperature (as a strategy of defence against hypoxia), previously known to occur in lower vertebrates, could also occur in mammals. Moreover, these studies pointed to thermal-control as one of the mechanisms possibly involved in hypoxic hypometabolism. Finally, the

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emerging notion that hypometabolism could occur in neonate mammals eventually offered an opportunity for new interpretations of the newbornâ&#x20AC;&#x2122;s decreased hypoxic ventilatory response to anoxia. The aim of this chapter is to examine the phenomenon of hypoxic hypometabolism in neonatal mammals, and its main implications. The interaction between metabolism and thermoregulatory and ventilatory responses will also be discussed.

2. Cerebral hypometabolism as a survival strategy for anoxic newborns Aerobic organisms depend essentially on oxygen as the final acceptor of electrons from oxidative metabolism, which is crucial for maintaining cell function due to electron flow through the respiratory chain connected with ATP synthesis. In this scenario, it is clear that hypoxia can put life of all aerobic organisms at risk.

In the first line of defence against parturitional anoxia cardiovascular adjustments play a crucial role. They consist of bradycardia and shunting of blood flow from peripheral tissue to central organs. Therefore, the organs such as the brain and heart are able to prolong remarkably aerobic metabolism under anoxic conditions [17].

Cell death caused by O2 lack begins when anaerobic ATP production fails to meet the energetic demands of ionic and osmotic equilibrium.

Immediately after the start of anoxia, cellular ATP levels begin to fall, and within 5 min of oxygen deprivation as much as 90% of the ATP is depleted in the mammalian brain [11]. The most significant ATP-related process in the majority of cells, and particularly in neurons, is ion pumping to maintain or reestablish transmembrane ion gradients. The sodium-potassium ATPase alone is responsible for 5 to 40% of cellular ATP turnover, depending on cell type. When only 50 to 65% of brain ATP is lost, membrane depolarisation occurs, and this causes the multiple negative consequences. Depolarisation results in a rapid influx of Na+ and water that is followed by an influx of Ca2+ through voltage-gated Ca2+ channels. The collapse of the sodium gradient causes the sodium-glutamate cotransporters activation to eject glutamate into the extracellular space where this neurotransmitter triggers a range of events, including activation of excitatory neurotransmission by stimulating N-methyl-D-aspartate (NMDA) receptors that are responsible for a significant part of the Ca2+ influx. The rise in free cytosolic intracellular calcium concentration results in the activation of Ca2+-dependent phospholipases and proteases, which further hasten the rate of membrane depolarisation, leading to uncontrolled cellular swelling, and ultimately to cell necrosis [3,4,10,18].

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If the restoration of oxygen occurs soon enough, these injuries caused by the low cellular ATP level can be reversible, but the reoxygenation of previously hypoxic tissues also stimulates a new set of injuries. The reoxygenation injury is caused by a burst of reactive oxygen species (ROS) generation, mainly superoxide radicals, from the highly reduced electron transport chain when oxygen is suddenly reintroduced. It can result in a direct damage by free radicals to macromolecules, including DNA, proteins, and membrane lipids. An indirect damage can also arise from inability of sarcoor endoplasmic reticulum membranes that are damaged by peroxidation to properly resequester Ca2+, thereby exacerbating the Ca2+-mediated damage caused by low energetics in the anoxic phase. Hence, anoxia-tolerant animals need to have mechanisms of adaptation that deal with the stresses on cellular energetics under anoxia. The above mentioned pathological sequence of events leading to cell death is the same in hypoxia-tolerant and hypoxiasensitive tissues, the difference being that the time course of the debilitating cascade in the former is counted in hours or days and in the latter in minutes [19].

There are two ways by which metabolic regulation can be applied to deal with the effects of low oxygen availability on cellular energetics [11]. The one is compensation, which works well in a short term, and the other is conservation. The first relies on increasing ATP production by oxygenindependent mechanisms, primarily utilizing the glycolytic pathway, in order to meet the demands of ATP-consuming cellular reactions. The second reduces the cell’s need for ATP supply. If ATP production is decreased due to low oxygen availability, then organisms turn down their rate of ATP consumption until a new rate of ATP turnover is established where ATP production again equals its consumption.

Profound reductions in ATPase activity during anoxia – a strategy widely used by anoxia-tolerant organisms – might at first glance be interpreted as the membrane pump failure. However, such decreases are brought about without any disruptions in electrochemical potentials, cellular ions or ATP concentrations [19,20,21]. Instead, the reduction in the Na+: K+ pump activity is thought to be a part of a highly coordinated process of energy conservation.

Moreover, a decrease in O2 availability initiates a generalized suppression of ion channels density and/or channel leak activities, which lowers the permeability of cell membranes and, therefore, the energetic costs of maintaining electrochemical gradients is minimized. This so-called ‘channel arrest’ phenomenon [18] is likely to be a key factor of the underlying mechanism of reduced ATP demand.

In models of anoxia-tolerant brain, the suppression of current flow through Na+ channels was observed, which led to the elimination of action

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potentials (‘spike arrest’), thereby reducing the energetic costs of neurotransmission and decreasing the ATP demands of the ion pumps involved in the maintenance of electrochemical gradients [19,20,21].

Regulating the demand for ATP during the transition to a hypometabolic state would seem to be the logical first step and it is supported by studies showing reduced protein synthesis, and Na+/K+ ATPase and NMDA receptor activities at the onset of anoxia [22]. The net effect of this balanced reduction of ATP supply and demand is that it spares fermentable fuel, reduces metabolic waste accumulation and extends survival time [19].

The majority of mammals, including humans, show poor tolerance to anoxia and their excitable cells and tissues are normally debilitated by any prolonged episode of O2 lack. As ATP generation by oxidative phosphorylation begins to fall off due to O2 limitation, the cellular ATP demands of most mammalian cells and tissues tend to remain constant, leading to an energetic deficit that can be compensated only by activation of anaerobic ATP supply pathways (the so-called ‘Pasteur effect’). The anaerobic ATP production can meet the sustained energy demands of the various cellular ATP consuming processes only temporarily. Moreover, limited stores of fermentable substrate together with the accumulation of deleterious endproducts (e.g. H+) exclude anaerobiosis as a long-term solution to severe oxygen shortage in these animals.

As was mentioned above ion-motive ATPases are likely to become the dominant energy sinks in anoxic mammalian cells, just as the Na+: K+ pump does in the anoxic turtle hepatocytes [19]. The important difference is that there appears to be little or no reduction in the absolute ATP demand of the ion-motive ATPases in mammals. Instead, anoxic mammalian cells show all the hallmarks of a non-adaptive ‘channel leak’ response. For instance, acute hypoxia increases the activities of Na+ - H+ and Na+- Ca2+ exchangers in rat neurons [23] implying an accelerated Na+ cycling, which might increase the preexisting ATP demand of ion-balancing ATPases. In addition, the catastrophic K+ efflux and associated collapse of membrane potential in anoxic/ischaemic mammalian heart and brain is thought to be caused by the opening of ATP-sensitive K+ channels [19], presumably also putting increased demands on Na+: K+ pump activity.

What we do not know at this stage is whether the enhanced anoxiadefence mechanisms of facultative anaerobes are unique, or whether they are simply exaggerated versions of processes still found to persist in mammals.

The strategy of large scale reduction in ATP demands does not seem to be necessary for newborns because of their lesser energy requirements. Oxygen tension in fetal brain is less than half of the normal value for adults. For example, the mass-specific cerebral oxygen and aerobic glucose consumption

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for the 7 day old rat is only about one-tenth that of the adult [3]. Thus the using up of brain ATP during anoxia is much slower in the neonate [1].

Without doubt a substantial part of the greater hypoxia tolerance of the neonatal brain can be attributed to its decreased energy demands.

The activity of creatine phosphokinase is correspondingly three to six folds less in neonatal brain [24] and the concentrations of mitochondrial enzymes for ATP production are also lower [25]. Another adaptive consequence of the lower rate of O2 consumption of the neonatal brain is a smaller blood-tissue O2 gradient compared with that in adults [26]. This would allow the neonate to have a lower systemic PO2 for the same tissue PO2 than that of the adult. However, the mitochondria of neonate and adult brain in situ appear to be similarly sensitive to reductions in blood oxygen content. Neonates show a critical decline in the mitochondrial phosphorylation potential at the same brain vascular oxygen saturation levels. In the neonate, however, this will occur at a lower arterial PO2 due to the higher oxygen affinity of neonatal blood [26]. In neonates, the size of the neurons is smaller, there are major changes in ion channel distribution and in receptor properties and there are far fewer synaptic connections, which is likely to have a strong influence on their hypoxia tolerance [27,28].

Other mechanisms of the neonatal anoxia tolerance may also be involved, for example the changes in activity of developmentally-regulated, oxygen-sensitive K+ channels, which regulates neuronal excitability [29,30,31]. A slow increase in extracellular K+ is followed by a fast rise which saturates at about 50-80 mM K+. In asphyxiated newborn mammals the pattern of cerebral changes in extracellular K+ is very similar to that seen in other hypoxia-tolerant vertebrates. The newborns require higher extracellular K+ levels to trigger the fast phase of the increase comparing with that in adults [32]. Moreover, the initial increase of cerebral extracellular K+ is also much slower in hypoxic newborn mammals comparing with that of adult brain [32]. Therefore, the time to reach the depolarisation triggering threshold is much longer in newborns. Studying hippocampal slices from young animals at the age of 30-40 days, Kass and Lipton [33] found that the evoked potential spike in granule cells of the dentate gyrus recovered 78% of its preanoxic amplitude after a 10 min anoxic episode. In the adult the recovery was only 4%. The authors linked the enhanced anoxic survival of the newborn to its greater ability to maintain ATP above a critical level of irreversible synaptic failure, which in turn seems to be independent of age.

Recently, Mohr et al. [34] proved that the main mechanism of hypoxic/ischemic resistance of the newbornâ&#x20AC;&#x2122;s brain is energy conservation, resulting in delayed loss of ATP, with a corresponding delay in glutamate release and non-glutamatergic damage pathways. Sodium channel down

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regulation – other protective mechanisms of survival discovered in turtle brain [35] – may also operate in the neonatal brain. Increasing the Na+ flux in cultured brain cells from the rat fetus produced a rapid down-regulation of the channels [36].

An anoxic-defence mechanism that mammalian cells share with their anoxia-tolerant lower vertebrate counterparts is the ability to reallocate energy between essential and non-essential ATP-demanding functions in the face of energy limitations. An active suppression of the N-methyl-D-aspartate receptor response to hypoxia-induced glutamate excitotoxicity [37], inhibition of gene transcription [38] and protein synthesis [39] leading to a shut-down of non-essential energy-consuming mechanisms interfering with cell survival – a process termed oxygen conformance – may also participate in mediating the early neuroprotective response observed during hypoxia in the newborns. In young animals the inhibition of cell repair, tissue growth and differentiation is observed, which contributes to the reduced VO2 [40].

In conclusion, functional analogies of the diminished cerebral vulnerability to hypoxia of the mammalian fetus with that of the turtle brain [17] do not seem to be the most important causes of the increased hypoxia tolerance of the neonate. The tolerance is rather a simple consequence of the comparatively undifferentiated state of the brain of the newborn, with its lower energy requirements and slower decline in ATP resources, lower excitability levels, delayed depolarisation and lesser scope to release harmful excitatory amino acids. Moreover, the neonatal brain shows also some “add on” features protecting it against hypoxia. These include enhanced levels of small protective molecules such as taurine as well as differences in the properties or regulation of ion channels and receptors [27]. Another compensatory mechanism seems to be anoxia-induced increase in mineralocorticoid receptors expression in the hippocampus, which limits damage to neurons [41]. All these factors increase tolerance of newborn mammals to anoxia.

3. Hypometabolism cooperates with anapyrexia in the protection against neonatal asphyxia It has been known for decades that body temperature decreases in rodents subjected to hypoxia, which extends survival in the hypoxic environment. In addition to the improved survival, it seems that the reduction in body temperature will protect the adult and neonatal mammals from the general pathological effects of hypoxia and hypoxemia.

Under hypoxic conditions adult rodents regulate their body temperature 3.1-6.0°C below the normoxic body temperature by means of both autonomic and behavioural thermoregulatory responses [42]. Moreover, in a variety of

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rodent species, such as rats, mice, hamsters and ground squirrels the thermoneutral zone has been reported to be shifted to lower temperatures during hypoxia exposure [42,43,44]. For example, rats housed in a temperature gradient and exposed to 6.9% oxygen for 6.5 hours quickly lowered their body core temperature from 37 to 34.5°C. This response was supported by a reduction in selected ambient temperature from 30 to 24°C. Replacing hypoxic air with normoxic air resulted in an increase in selected ambient temperature, which was followed by rapid return to the normothermic core temperature [45]. The hypoxic rat seems to modulate both behavioural and autonomic thermoregulatory responses to reduce its body temperature for at least six hours of hypoxia. Because animals exposed to the insults selected cooler ambient temperatures and maintained the decreased body temperature, it would be concluded that the set-point for control of body temperature is reduced during and after the exposure to the insult [46,47].

Such a regulated decrease in the thermal set-point is referred to as anapyrexia [48,49]. Anapyrexia is the most efficacious mechanism of physiological defence against neurotoxicity induced by hypoxia [13,47,50]. Many ectothermic organisms (including the unicellular Paramecium caudatum) also seek cooler ambient temperatures when challenged by hypoxia and thereby use a decrease in body temperature to reduce tissue demands for oxygen [11,51]. The argument that anapyrexia increases survival of hypoxic animals, together with the fact that this response is extremely widespread among taxa, provides evidence that it is an adaptive response.

Besides their ability to use anapyrexia [52] newborn rats maintain their core temperature at 32-33°C (which is 4-5°C lower than that in adults) [52,53]. The reduced body temperature of newborns is often regarded as a sign of immaturity of their thermoregulatory system [17,53]. Neonates of altricial rodents exhibit only a limited ability to thermoregulate and they become fully homeothermic at an age of approximately 20-30 days, when their body temperature reaches its normal value [53,54]. In their nest, however, they can maintain their physiological body temperature of 32-33°C, at an extremely low ambient temperature of 13°C as well as at 25°C [52]. Their body temperature remains markedly lower than that of adults even during a permanent exposure of the newborns to 35°C, which is the highest ambient temperature they can tolerate permanently [55].

Therefore, the reduced body temperature of newborn rats should not be referred to as hypothermia. It is their physiological body temperature. This particular level of thermal regulation must have evolved to protect the newborns against the detrimental effects of parturitional asphyxia.

Surprisingly, in mice anapyrexia develops already in fetuses in utero just before labour [47].

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In addition to the physiologically reduced body temperature newborn rodents develop anapyrexia in response to hypoxia [52]. The 2-day-old guinea pigs in10% oxygen decrease body temperature and choose an ambient temperature lower than that in normoxia [56]. Behavioural responses of rat pups to cold, such as reduction of the exposed body surface and huddling, are also depressed by hypoxia [57]. Most or all of these changes can be interpreted as a lowering of the set-point of body temperature regulation, which is a sine qua non condition for hypometabolic responses in mammals.

In the hypoxic newborn, the artificial increase of body temperature to the normoxic value evokes responses like hyperpnoea and a drop in systemic vascular resistance, which indicates the newbornâ&#x20AC;&#x2122;s attempt to dissipate the heat excess. Such a forced clamping of body temperature at its normoxic value might be interpreted as a relative hyperthermia, which would be counterproductive for the delivery of oxygen to the essential organs [40].

There is a clear-cut difference between anapyrexia and hypothermia [12,44,46,58]. In mammals, anapyrexia is not accompanied by a thermogenic and oxygen consuming responses, which is quite opposite to hypothermia, emphasizing the difference between anapyrexia and hypothermia [49].

The thermoregulatory set-point is established as a balance between warm-and cold-sensitive neurons located in the preoptic region of anterior hypothalamus (POAH) [59]. Both fever and anapyrexia are brought about by shifts in the thermal balance as a result of changes in the hypothalamic temperature thresholds for activation of autonomic and behavioural thermoeffectors [47,59]. There is a growing body of evidence that the drop in body temperature caused by hypoxia is a consequence of a downward resetting of the thermoregulatory set-point [13,42,44,45,47,50].

Anapyrexia occurs when internal and/or external factors reduce the setpoint of temperature below the normal body temperature. The hypothalamic center responds by activating thermoeffectors of heat loss and inhibiting thermoeffectors of heat production. These responses persist until core temperature reaches the new set-point temperature [46].

The preoptic area and anterior hypothalamus (PO/AH) are recognized as the major integrative center for thermoregulation, and it is likely that neurotransmitters released by hypoxic stress may act there to reduce body temperature. Recently, adenosine, serotonin, nitric oxide (NO), carbon monoxide (CO) and arginine vasopressin have been suggested as putative mediators of anapyrexia [13,50]. In addition, hypoxia has been shown to activate dopaminergic systems in the central nervous system [60] and to induce c-fos in the anterior hypothalamus [61]. Actually, dopamine is one of the most important thermoregulatory neurotransmitters, and it is accepted that

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the increase in dopamine concentrations in the preoptic area causes reduction of body temperature in mammals [62]. The preoptic area is a key site for dopamine action in the regulation of body temperature during normoxia (through D2 receptors) and hypoxia (through D1 receptors) [63].

Hypoxia-induced anapyrexia results partially from the impairment of oxidative phosphorylation in the CNS [64], and depletion the brain of glucose, which impairs oxidative phosphorylation by reducing availability of the metabolic substrate [65]. Taken together, these results imply that a reduction in oxidative phosphorylation in the CNS is important for the development of anapyrexia.

It has been suggested that the inhibition of cutaneous vasoconstriction [66] and/or inhibition of shivering [40] and non-shivering thermogenesis [67] take part in the thermoregulatory response to hypoxia. Indeed, peripheral chemoreceptor-evoked inhibition of brown adipose tissue (BAT) sympathetic nerve activity was reported to directly contribute to the hypoxic anapyrexia [68]. The hypoxic atmosphere also causes a transient increase in dry heat loss and reduction in CO2 production, reflecting peripheral vasodilatation and reduced metabolism during the initial stages of hypoxia [45].

Hypoxic anapyrexia is accompanied by (i) a decrease in metabolic rate [12], resulting in an inhibition of thermogenesis [44], (ii) an attenuation of ventilation and cardiac activity [14,52,69], (iii) an increased O2 affinity of hemoglobin improving O2 extraction in the lungs [70], (iv) an increase in heat loss [58] and (v) an enhancement of survival rate [51,52,70]. Coexistence of hypometabolism and anapyrexia might suggest that hypoxia inhibits that component of normoxic oxygen consumption which concerns heat production. In newborns, however, hypoxic hypometabolism commonly occurs even at thermoneutrality. This means that the hypoxic inhibition affects some oxygen-dependent functions, other than heat production [71].

Several considerations really indicate that hypometabolism is not simply an expression of oxygen limitation, nor is the consequence of the drop in body temperature (Q10 effect). The most important finding was that the drop in VO2 precedes, not follows, the decrease of body temperature, indicating that the latter can not be the cause of the former. In addition, the magnitude of the hypoxic drop in body temperature (1–3°C) is too small to explain the metabolic drop as a Q10 effect [71].

The fact that even mild reduction in brain temperature can provide a significant reduction in brain injuries [72] suggests that they might not rely on reduction in metabolic rate only. When cerebral metabolic rate is reduced correspondingly by drugs, this does not provide the same neuroprotection [73]. Even if the detailed mechanisms responsible for the anapyrexia and the drop in VO2 during hypoxia are not clear, there is no doubt that the following

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two processes reinforce each other in the protection against hypoxia: the decrease in VO2 contributes to the drop in body temperature, and the latter, in turn, reduces VO2 below its normoxic level [71]. In conclusion, the hypoxic decrease in body temperature and the drop in VO2, reflect two controlled processes, which are likely to be coordinated between, but not causative of each other.

Nowadays the neuroprotective role of anapyrexia is commonly known. It favours hypoxic survival in animals of many classes, ranging from lower vertebrates to unicellular organisms. With respect to neonatal mammals, the importance of the reduced body temperature during hypoxia has been confirmed repeatedly since the earliest studies of the topic [15,74], and has been documented experimentally many times thereafter. Posthypoxic decrease in body temperature is neuroprotective in the neonatal rat [75,76] and newborn pig [77,78]. Animals such as the rat or dog pups, that are immature at birth, increase their anoxic tolerance from 5 min at 37째C to 40 min at 15째C [74]. Thoresen et al. [79] showed for the first time in a randomized study on 7-day-old neonatal rats that cooling initiated after the injury uniformly protected all areas of the brain. The pups were cooled from 38 to 32째C for 3h, reaching target temperature within 15 minutes.

Histological evaluation of the injury, performed one week later, showed more than 50% protection.

Anapyrexia has multiple neuroprotective effects, including reduction of cerebral demand for oxygen [80], prolonged delay of a spread depolarisation of cerebral neurons [81], prevention of massive calcium entry into neurons [82], suppression of both excitotoxic glutamate release [83] and its neurotoxicity [34,84], reduction of free radical formation [83,85,86], prevention of brain edema [87], and reduction of hemorrhage and neutrophil infiltration [88]. Moreover, the naturally reduced neonatal body temperature prevents anoxic acidosis and hyperferremia [89]. The reduced temperature diminishes lipid peroxidation [86] and protects ischemic cell membranes against potassium efflux [90]. In addition, mildly decreased body temperature can reduce the activation of cytokine and coagulation cascades by increasing concentrations of interleukin-10 (IL-10, an antiinflammatory cytokine) and reducing concentrations of tumor necrosis factor alpha (TNF-a) through increased activation of suppressor signaling pathways and by inhibiting release of platelet activating factor [91]. Anapyrexia helps maintain cerebral metabolism both during and after cerebral insults. It also prevents or ameliorates secondary cerebral energy failure, which preserves cerebral highenergy phosphates [92] and prevents the secondary free-radical damage [93,94,95] to the brain.

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A mild decrease in body temperature can also help to reduce the number of cells undergoing apoptosis after hypoxia/ischemia [96]. In hypoxic rats the drop in body temperature has been shown to come into effect through inhibition of caspase activation, thereby preventing apoptotic cell death [97].

Experimental and clinical studies indicate that the number of apoptotic (but not necrotic) neurons is reduced, caspase activity is lessened, and cytochrome c translocation is diminished by the mild decrease in body temperature [96,97]; there might also be an increase in expression of the anti- apoptotic protein Bcl-2 [98].

How does the decreased body temperature provide neuroprotection?

First, it seems clear that a lower temperature protects tissues deprived of oxygen by slowing the rate of cellular damage that occurs from formation of free radicals, chemical metabolites, and tissue edema. The absorption, distribution, metabolic activation and deactivation, and excretion of a chemical involve biochemical reactions that are temperature-dependent.

Secondly, anapyrexia exerts its protective effect by a leftward shift in the oxyhemoglobin dissociation curve with the increase in oxygen loading in the lungs as a result. And thirdly, both the ventilatory and cardiovascular responses to hypoxia, which are oxygen consuming, are decreased at lower body temperatures [14,52].

It must be stressed, that the effects of anapyrexia are long-term and persistent. Decrease in body temperature of asphyxiated newborn rats not only significantly increases survival, but also results in unimpaired motor as well as improved cognitive functions [99,100]. The reduced body temperature prevents postanoxic disturbances, such as abnormal responses to stress and memory impairment in rats from their youth up to senescence [101,102,103,104].

In addition to the direct cerebroprotective effects mentioned above, anapyrexia delays the onset of energy failure and, therefore, the therapeutic time-window for neurological treatment of asphyxiated newborns is markedly prolonged. During anoxia, cerebral neurons start being injured when ATP resources are reduced to less than 25%, however at 0–10% irreversible neuronal death occurs. These critical levels are similar for all species and ages, what differs is the time to the critical energy depletion and spreading depolarisation in the brain. Some neurons are irreversibly damaged to the point where nothing can be done; however, there are multiple factors determining whether neurons that are reversibly injured go on to die or survive [28]. Whole-body cooling for 24 hours at either 35°C or 33°C commenced 2 hours after resuscitation of asphyxiated piglets prolonged the therapeutic time-window and reduced the overall secondary energy falls during 48 hours after hypoxia/ischemia [92]. Thus anapyrexia extends the

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window of opportunity to reduce, or even halt the cascade of damaging events, and hence rescue the neurons that have been reversibly injured after the acute insult. It exerts protection by interfering with several steps in this cascade, being protective in itself as well as prolonging the time-window by which other neuroprotective strategies, e.g. antioxidant and antiapoptotic drugs, may be applied [28].

4. Therapeutic cooling of asphyxiated infants Clinical interest in decreasing body temperature began in the 1950s with some case reports of successful resuscitation after immersing asphyxiated infants in a tub of cold water [105,106]. These early studies also showed that babies vary in their temperature requirements according to their health status [107]. Indeed, recent clinical studies [108] show that the range of ambient temperatures at which babies start sweating to lose heat is surprisingly wide (33 – 38°C). This suggests that there are substantial differences in the setpoint of temperature regulation in the infants.

The phenomenon of decreased body temperature was described more than 50 years ago in small preterm babies as well as in term babies suffering from perinatal asphyxia [105,106,109]. In small preterm babies receiving conventional care deep body temperature is frequently less than 36°C in the first week of their life and it is not always connected with any sustained increase in heat production [109]. This proves the existence of the regulated decrease of body temperature in preterm newborns during first days of their life. Burnard and Cross [105] compared babies suffering from asphyxia (defined as a failure to establish respiration within 3 min of birth) with nonasphyxiated babies. The asphyxiated infants maintained their rectal temperature at about 34.5°C within 16 h postpartum while the control babies were ~ 2°C warmer. The authors suggested that this natural decrease of body temperature might have a survival value. However, this concept was abandoned in the1960s with the report of increased mortality of hypothermic preterm infants [110].

Accordingly, the adaptive thermal responses of asphyxiated human newborns have been lost from the clinical research for four decades. In neonatalogical practice asphyxiated preterm infants are commonly being forced to stabilize their body temperature at 37ºC by means of incubators.

The results obtained recently [111,112] confirm those from the early investigations [105]. Infants with moderate-to-severe encephalopathy were randomized to receive either conventional care or therapeutic decrease in body temperature. Infants in the conventional care group were kept at an ambient temperature of 25 – 26°C and maintained their axillary and rectal temperatures at 32.5 -33°C for up to15 h after birth despite using standard

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rewarming methods such as swaddling or placing rubber gloves filled with warm water around them [111]. In the second paper [112] asphyxiated babies were given an opportunity for passive cooling. Most of the newborns (89%) achieved the target body temperature within the range of 33.0 - 34.0°C. These data focused again neonatologists’ attention on the phenomenon of regulated decrease in body temperature in asphyxiated infants.

Cooling applied both during and after hypoxia-ischemia seems to be the most efficacious protective strategy because of multiple effects at different levels within pathways that contribute to brain injury after the insult [113].

Within the last decade, therapeutic cooling for infants suffering from perinatal asphyxial encephalopathy has been applied in preclinical models [76,77] and there are several major randomized clinical trials in the developed world [114,115]. These clinical trials confirmed that selective head cooling may prevent encephalopathy in term infants at the highest risk for perinatal hypoxic-ischaemic brain injury [114]. The efficacy and safety of whole-body cooling initiated within 6 hours of age to a depth of 33.5°C for 72 hours duration has also been demonstrated [116]. Selective head cooling combined with mild systemic decrease of body temperature for 72 hours after hypoxia/ischemia significantly decreased the combined outcome of severe disability and death of newborns [117]. The aim of selective head cooling is to achieve adequate cerebral cooling with only a small reduction in core body temperature, thus minimizing the potential harmful effects of cooling [118].

It must be stressed that the cooling has not been associated with an increased risk of infection. Therefore, the preliminary studies suggest that therapeutic cooling could be carried out safely and this was confirmed in large randomized controlled trials [118].

The extent of neuroprotection achieved by decrease in body temperature depends on the time of initiation of the cooling and its duration. Prolonged cooling within the “therapeutic window” (defined in the current neonatal trials as within 6 h since birth) is essential for long-term, successful protection [119].

Cooling device should induce cooling rapidly to the target temperature (core temperature of 33 – 34°C for whole-body cooling, [115,120] or 34 – 35°C for head cooling [114,120] and maintain the core temperature tightly within the target range for the desired duration (usually 72h) and should allow rewarming in a slow and controlled manner at a set rate (usually at 0.2 -0.5°C/h) [121]. It should be stressed, that the earlier cooling is commenced the better the outcome is [119]. This is particularly critical because the therapeutic window is substantially reduced with increasing severity of injury [120].

Avoidance of overshoot hyperthermia is essential during rewarming; the brain is extremely sensitive to small changes in temperature and in animal

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studies even mild hyperthermia worsened all aspects of the neurotoxic cascade [122,123]. Indeed, artificial warming, forcing the body temperature of a hypoxic newborn to rise to the normoxic value, provokes energydemanding physiological responses counterproductive to survival. Among these, a decrease in peripheral vascular resistance (a demonstration of the newborn’s attempt to dissipate heat by diverting blood to the body periphery) obviously hindering O2 delivery to the core organs [124] seems worth mentioning. In our previous work we showed that in newborn rats exposed to anoxia at physiological body temperature of 33°C anapyretic drop in body temperature was the most efficacious and continued over more than 10 min postanoxia. This, however, was prevented under external conditions necessary to clamp body temperatures at 37°C or 39°C. Clearly, thermolytic responses of newborns exposed to the hyperthermic conditions must have reached their maximum capacity already prior to anoxia and they were unable to develop the anapyretic response [52]. During hypoxia at an ambient temperature of 34°C mice maintain their normal body temperature but they become anapyretic at the cooler ambient temperatures, which results in enhanced tolerance to hypoxia [46].

The early studies of Hey [109] confirmed that mortality rises if deep body temperature is allowed to approach 37.8°C in babies weighing <2 kg at birth. Recent clinical trials also show that relatively high temperatures applied in incubators during usual care following hypoxia-ischemia is associated with increased risk of adverse outcomes [108]. Infants developing fever of 38ºC or more show an elevated rate of unfavorable outcomes. In addition, maternal fever during parturition is associated with a greater likelihood of a low 1 minute Apgar score in newborns, with disturbances of their respiration, and with the need for bag-mask ventilation in the delivery room [125]. A case-control study design demonstrated that intrapartum fever, which was unlikely to be infectious in origin, was associated with a 3-4-fold risk of unexplained early-onset seizures in term infants [126].

Given the importance of the low body temperature in hypoxia, it may be asked whether or not its further reduction by external means, to values even lower than those naturally attained, may be beneficial. In ectotherms, body temperatures lower than that determined by the ambient temperature chosen by the hypoxic animals are likely to be protective [71,80]. Small newborn mammals are often born altricial, with an ectothermic-like behaviour. Also in these cases, cold exposure, below the physiological level might have a favourable effect on the survival in hypoxia. For example, in newborn rats with hypothermic body temperature of 31°C, the successful respiratory activity lasted to the end of scheduled time of anoxia (25 min) as in pups with physiological body temperature of 33°C [52]. Nevertheless,

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hypothermia induced metabolic acidosis under normoxic conditions, although the arterial blood pH was hardly influenced by anoxia [89].

Forcing decreases in body temperature below its regulated level (i.e. below the set-point of body temperature) will evoke different physiological responses compared with those accompanying the regulated reduction. The manner by which temperature is reduced will have a marked impact on the animalâ&#x20AC;&#x2122;s health and general physiological state. Moreover, forcing the reduction in body temperature below the value naturally attained can stimulate heat production, which is not a desirable event in hypoxic conditions [71]. The greater the decrease in body temperature, the greater the increase in the error signal in the thermoregulatory centre, resulting in more efficacious thermogenic responses [46]. Cold-stressed newborn infants increase heat production by non-shivering thermogenesis without any increase in physical activity. Non-shivering thermogenesis occurs in brown adipose tissue (found in newborns between the scapulae and along the aorta) by oxidizing fatty acids in the mitochondria to produce heat [127]. In newborn infants in response to cold also occurs peripheral vasoconstriction, diverting blood away from the body surface to the core.

From a clinical perspective, these physiological responses would be considered undesirable and would complicate a clinical treatment.

Excessive body cooling activates the thyroid and adrenal systems and influences immune responses, lipid hydrolysis, mobilisation of free fatty acids, glucoregulation, and results in tachycardia, tachypnea, and altered renal, hepatic, and gastrointestinal functions as well [46]. It means that there is an enhanced risk of systemic complications when cooling is excessive (<30°C), which might counteract the potential benefit of the cooling [120].

Important associations between reduced or elevated body temperature at birth and subsequent morbidity and mortality [51,52,76,92,94,95,99,100,108,110,114,115,116,117,122,123,125,128] suggest that upward shifts in body temperature at birth due to hyperthermia or fever, are neurotoxic while downward shifts, due to anapyrexia or hypothermia, are neuroprotective. The relationship is shown in Figure 1. Asphyxiated human newborns should be moderately cooled to prevent not only early neurological deterioration, which has been shown in 12-months follow-up [119,129] but also a variety of much more delayed disturbances such as attention deficithyperactivity disorder (ADHD) in childhood [130].

Despite the clinical heterogeneity of perinatal asphyxia and the use of different cooling methods, there are consistent findings that the decrease in body temperature reduces the extent of neurological damage and improves survival without disability [128]. Therapeutic cooling is now widely offered

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Figure 1. Effects of anapyrexia or hypothermia (left side of the diagram) and hyperthermia or fever (right side of the diagram) upon neurotoxicity induced by parturitional asphyxia.

to moderately or severely asphyxiated infants in countries and centers which participated in the trials [131]. At present, however, the therapeutic decrease of body temperature in low-resource and transitional-care countries has not been allowed [111].

5. Modulated ventilatory response to anoxia in newborns For many years neonatologists have been deeply puzzled by the fact that newborn infants presented a decreased ventilatory response to hypoxia. This phenomenon becomes clear when it is considered in light of the metabolic changes [132]. This ventilatory decline is associated with the reduction of body temperature and metabolic rate [44]. Exposure of newborn mammals to anoxic conditions elicits a characteristic pattern of respiratory responses [52,133,135]. Immediately after the beginning of the exposure the initial, transient hyperpnoea occurs and is followed by primary apnoea. Then the gasping response emerges. In developing rats it exhibits a triphasic pattern.

The first phase of gasping is characterised by rapid gasps. The subsequent, much longer second phase of extremely slow gasps is replaced by the third phase of rapid respiratory efforts that eventually wanes and gives way to the terminal apnoea and death [133,134,135,136].

Differently from the hypoxic hyperventilation, which is evoked by peripheral chemoreceptors stimulation, the hypoxic ventilatory decline is mediated directly by the central nervous system [137]. Considering the interaction between respiratory and thermoregulatory functions during hypoxia the authors pointed at hypothalamus (exactly anteroventral preoptic region of

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the hypothalamus - AVPO) as an important brain area that plays a role in the control of body temperature and ventilation [138]. There is a growing body of evidence that the key mediator that plays a role in the hypoxic ventilatory depression as well as in anapyrexia is adenosine [13,50,139]. Adenosine (a purine nucleoside) is a neuromediator involved in many inhibitory cerebral mechanisms and thus it reduces cerebral metabolic rate. Intracerebral concentrations of adenosine rise during hypoxia and are associated with increases in local cortical blood flow, decreases in whole-body oxygen consumption, reduction in body temperature and protection against cerebral damage [13,50,140]. Recently Barros et al. [13] provided new evidence that adenosine acts in the AVPO, affecting processes underlying the maintenance of body temperature in euthermia, and influences the hypoxic ventilatory response through A1 receptors. Fewell et al. [141] have provided additional data that adenosine, acting via adenosine A1 receptors, plays a role in modulating hypoxic gasping and in mediating the profound bradycardia, which occurs coincident with hypoxia-induced primary apnoea in rat pups during the first 10â&#x20AC;&#x201C;11 days of postnatal life. Adenosine functions as a regulatory hormone increasing cardiac oxygen delivery and decreasing cardiac work during periods of limited oxygen supply [142,143]. In the rat pup the adenosine-mediated bradycardia is likely to play an important role in tempering the work load imposed on the heart during hypoxia and catecholamine stimulation, thus protecting the myocardium against the adverse consequences of decreased oxygen supply [142,143]. With regard to survival, adenosine modulation of hypoxic gasping may offer some advantage by prolonged stimulation of a slow pattern of gasps until oxygen becomes available.

Platelet-activating factor (PAF) has also been regarded as a modulator of ventilatory and metabolic responses to hypoxia [144]. Platelet-activating factor receptors (PAFR) are constitutively expressed in the pontomedullary regions in relative abundance suggesting functional importance in this area [145]. PAFR activation selectively mediates important components of the ventilatory and metabolic responses to hypoxia, and does not appear to be involved in central chemosensitivity. It is possible that imbalances in PAFR activity may lead to maladaptive regulation of the tightly controlled relationships between ventilatory and metabolic pathways during hypoxia [144].

The initial respiratory response of newborn mammals to anoxia is hyperpnoea [146] combined with arousal which precedes primary apnoea and profound bradycardia. During the arousal phase, the pup is awake and exhibits pronounced locomotor activity in an apparent attempt to escape from the anoxic environment. Tonic posturing with the neck and back arched and extremities extended (opisthotonos) appears prior to the onset of primary

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apnoea [141]. The apnoea during maintained lung inflation (Hering-Breuer inspiration-inhibitory reflex) is an index of the vagal inhibition of breathing that originates from the lungs during inspiration. The strength of this reflex decreases in adult animals under hypoxic conditions, because the activation of the chemoreceptors, stimulating breathing, offsets the vagal inhibition.

However, in 2-day-old rat pups, this is not so; contrary to what happens in older animals, the Hering-Breuer reflex remains unaltered, or even increases during hypoxia, compared to normoxia [147]. This phenomenon is interesting in the light of hypoxic anapyrexia and hypometabolism described above. The possibility exists that in the acutely hypoxic newborn the decrease of the metabolic stimuli enhances the efficacy of the inhibitory inputs, with the potentials for creating a vicious cycle further inhibiting breathing [71]. During primary apnoea, the pup becomes limp and unresponsive to somatosensory stimuli before the onset of gasping. It might represent a period necessary to switch from neural mechanisms of regular breathing to those governing gasping.

A multiphasic gasping pattern – as first described by Selle and Witten in 1941 [141] – persists in rat pups up to ~20 days of postnatal age and is followed by a monophasic gasping pattern in older animals [133]. In hyperthermic (37°C) 2-day-old rat pups we were able to observe all phases of gasping. Primary apnoea was followed by a period of rapid gasping that lasted ~2 min (phase I of gasping); this period of rapid gasping was followed by a period of slower gasping (one to two gasps per minute) that lasted for ~8min (phase II); finally there was a period of rapid gasping (~4min, phase III of gasping) which eventually waned and gave way to secondary or terminal apnoea [52]. The gasping phase is characterized by a generalized loss of aerobic body function and of electroencephalographic activity producing a state of lathergy [148]. At this point, the pup is unresponsive and such a state is called “asphyxial coma”.

Gasping is the most interesting respiratory response of newborns to anoxia, enabling them to autoresuscitate when oxygen is made available [149]. The neural substrate of gasp generation is the lateral tegmental field of the medulla. Some authors suggest that there is the possibility that the critical regions for respiratory rhythmicity may dynamically reconfigurate to generate gasping [150]. Gozal et al. [134] and Gozal and Torres [151] have shown that nitric oxide – a signalling molecule that influences neuronal excitability – plays a role in initiating and modulating the pattern of gasping in rat pups during exposure to anoxia. Their experiments have shown that nitric oxide favours the early appearance of gasps, increases gasp frequency and limits anoxic tolerance during exposure to anoxia [134,151]. Expression of neuronal nitric oxide synthase and its activity enhances with increasing postnatal age within the neural sites responsible for gasp generation and

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underlie some of the characteristic developmental changes in gasp activity [134,151]. Moreover, N-methyl-D- aspartate glutamate receptors are critically involved in particular components of gasp maturation [136], specifically in the early phases of respiratory activity that follow primary apnoea. An administration of MK 801 (a non-competitive NMDA glutamate receptor channel antagonist) prolonged the duration of primary apnoea, increased the time to terminal gasp, and altered all three phases of gasping.

Thus, it seems that glutamate and nitric oxide (cerebral concentrations of both of them are increased during hypoxia) [151] can influence the initiation of gasping following primary apnoea during anoxic exposure. In addition, an intact adrenal gland is required for a normal gasping response during anoxia in 1-and 8-day-old rat pups [152].

If oxygen becomes available during gasping following hypoxic-induced apnoea, recovery is possible. The process of recovery from hypoxia-induced apnoea by gasping was first termed “self-resuscitation” by Adolph in 1969 and subsequently termed “autoresuscitation” by Guntheroth and Kawabori in 1975 [141]. Successful autoresuscitation from apnoea requires integration of several physiological systems including the nervous, respiratory and cardiovascular systems. There are sequential cardiorespiratory stages of a successful autoresuscitation: first, introduction of air into the lungs by gasping; second, transport of oxygen from the lungs to the heart; third, increasing heart rate and cardiac output; and fourth, responses of the nervous system to reoxygenation and increased perfusion [153].

As early as in 1972, Morgan and Guntheroth [141] speculated that the failure of autoresuscitation from apnoea might lead to some cases of sudden infant death syndrome (SIDS). In the last decade, the incidence of SIDS has decreased, primarily as a result of public campaigns aiming to reduce prone sleeping position in babies [154]. However, SIDS still remains the major cause of death in apparently healthy infants. Although the precise mechanisms underlying SIDS remain undefined, one of the leading hypotheses posits that recurrent episodes of hypoxemia may precede the fatal event and affect gasping and autoresuscitation mechanisms [155]. Interruptions of ventilation or apnoea occur frequently in premature [156] and term [157] newborns as well as in older infants during sleep, thus impairing the uptake of oxygen and removal of carbon dioxide. In human newborns, spontaneous recovery from sleep-related apnoea (central, obstructive or mixed) or positional asphyxia can occur early with or without behavioural [158] and/or EEG arousal [159] or later as a result of autoresuscitation from “asphyxial coma” by hypoxic gasping [159]. Because the coma occurs when early defence mechanisms are absent or fail to resolve apnoea or positional (facedown) asphyxia, gasping is considered to be the last operative mechanism used by mammals to ensure survival during exposure to severe hypoxia.

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Reports by Poets et al. [155] and Sridhar et al. [160] have documented failure of hypoxic gasping to initiate autoresuscitation and prevent death in a number of SIDS infants. The majority of these infants displayed stage I of autoresuscitation (i.e., gasping), but gasping failed to produce stage II of autoresuscitation (i.e., cardiac resuscitation with a rapid increase in heart rate).

The understanding of the physiological mechanisms of autoresuscitation success/failure from animal studies would provide an objective basis for understanding mechanisms of autoresuscitation failure in humans. A number of factors have been shown to influence the onset, duration and number of gasps during exposure to hypoxia in newborn animals and infants. Both brain anoxia and stimulation of the carotid chemoreceptors are necessary to induce gasping in rats and the response is terminated by “central” failure as the carotid receptors discharge long after gasping ends [141].

One of the most important factors which influence the pattern of respiratory activity during anoxia is body temperature. The duration of the hyperpnoea and apnoea was inversely proportional to body temperature [52].

The longest period of hyperpnoea (52±4 s) was recorded in newborns exposed to anoxia at 33°C, which confirms that at this particular, physiological body temperature oxygen consumption is the lowest.

Body temperature influences also the duration of the subsequent gasping phases. The frequency of the most quiescent (phase II) gasping at physiological body temperature of 33°C was as low as 0.6±0.04 gasps/min [52]. Such a low gasping frequency might provide newborns sufficient control over oxygen availability in air at a minimum energetic cost. The gasping frequency raised under hyperthermic conditions. It must be stressed that anoxia tolerance in newborn rats is highly temperature-dependent and the time to the critical gasping phase corresponds to anoxic survival time [52,133]. The maximum tolerance time for newborn rats is 50 min [161].

Because at the two lowest body temperatures (31 and 33°C) rat pups kept on gasping phase II till the end of 25 min period of exposure to nitrogen they were far from the critical anoxia. In contrast, newborns at body temperature of 39°C reached the critical anoxia within 9 min exposure to nitrogen [52].

Neonatal anoxia under hyperthermic conditions alters the early postnatal (developmental) profile of plasma corticosterone in 14 days old rats. Neonatal anoxia combined with hyperthermia decreased the corticosterone response to open-field stress. The basal values of corticosterone in animals exposed neonatally to hyperthermia both anoxic and control ones tended to be smaller as well [162]. The deficit of the corticosteroids may retard appropriate lung maturation, thus it is possible that reductions in plasma corticosterone persisting over the first weeks of life could contribute to early respiratory morbidity. Extensive epidemiological studies have identified the major risk

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factors for sudden infant death syndrome, which included hypoxia and apnoea and extreme alterations in body temperature [163]. Low night-time cortisol level, associated with changes accompanying development of adult-type nighttime temperature rhythm, was proposed as a ‘window of vulnerability’ to SIDS [163]. These data confirmed the previous findings that the association between hypoxia and hyperthermia seems to be responsible for the cascade of events leading to neonatal apnoeas and sudden infant death [164,165]. All mechanisms used to cope with parturitional asphyxia are summarized in Table 1.

Table 1. Time-course of start and continuation of mechanisms involved in cerebral defence against parturitional asphyxia.

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6. Conclusions The substantial part of the greater hypoxia tolerance of the neonatal brain can be attributed to its lower energy requirements. They are a simple consequence of the comparatively undifferentiated state of the newborn’s brain. Moreover, newborn mammals have a unique combination of some “add on” physiological features to cope with parturitional asphyxia. They maintain markedly reduced body temperature and they display a remarkable gasping ability under anoxic conditions. In addition to the physiologically reduced body temperature newborn rodents develop anapyrexia (regulated decrease of thermoregulatory set-point) in response to hypoxia. Anapyrexia has got multiple neuroprotective effects and favours hypoxic survival in newborns.

The reduced temperature can prevent cellular damage by maintaince of hypometabolic state. This means that a reduction in temperature will lead to a reduction in the rate of cellular respiration, oxygen demand or carbon dioxide production. The reduced demand for oxygen accompanying anapyrexia is especially critical in highly aerobic organs, such as the brain and heart, subjected to hypoxia/ischemia. Anapyrexia provides neuroprotection by interfering with several steps of pathological cascade under hypoxic conditions. From clinical point of view the most important neuroprotective effect of reduced body temperature is the ability to prolong the time-window by which other therapeutic strategies, e.g. antioxidant and antiapoptotic drugs, may be applied.

Nowadays the neuroprotective role of anapyrexia is commonly known.

Important associations between reduced or elevated body temperature at birth and subsequent morbidity and mortality are proved [108]. They suggest that shifts in body temperature at birth cause brain damage (hyperthermia) or have a neuroprotective value (anapyrexia).

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates, 2011: 147-182 ISBN: 978-81-308-0471-2 Editors: Anna Nowakowska and MichaĹ&#x201A; Caputa

7. PI3K-Akt regulation as a molecular mechanism of the stress response during aerobic dormancy Jing Zhang, Shannon N. Tessier and Kenneth B. Storey

Institute of Biochemistry & Department of Biology, Carleton University, 1125 Colonel

By Drive, Ottawa, Ontario, K1S 5B6, Canada

Abstract. The biochemical mechanisms that direct intracellular signal transduction networks have been extensively studied in a variety of organisms spanning invertebrates to vertebrates.

Depending on environmental conditions, animals may adjust these cellular pathways in favour of growth signals or, at the opposite extreme, initiate death signals. Animals living in extreme environments are often faced with stresses (e.g. oxygen, water and/or fuel limitation, temperature extremes) and as a result have evolved adaptations that ensure safe transitions into hypometabolic states (e.g. dormancy, hibernation, aestivation, diapauses, etc.) when conditions are unfavourable for normal life. Metabolic rate may be reduced by as much as 70-99% as compared with active states and a variety of preservation strategies may be activated, all relying on the restructuring of evolutionarily conserved metabolic pathways. Akt/PKB is a pivotal stress kinase which exerts control over pathways including glucose metabolism Correspondence/Reprint request: Dr. Kenneth B. Storey, Institute of Biochemistry & Department of Biology Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, K1S 5B6, Canada E-mail: kenneth_storey@carleton.ca

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(through GSK-3), protein synthesis (through mTOR), cell cycle regulation (through p27KIP1 and MDM2), and apoptosis (through BAD and caspases). In addition, via cross-talk with the NF-κB, Wnt, and MAPK signalling pathways, Akt/PKB is placed in a unique regulatory position in the stress response. By evaluating the natural responses of Akt/PKB signalling in diverse animal groups facing a range of stress challenges, researchers will discover the cellular plasticity of central metabolic pathways, how these pathways may be altered depending on the needs of the organism, and finally unravel important regulatory secrets involved in hundreds of stress and disease pathologies.

List of symbols and abbreviations 4EBP Apaf-1 ASK1

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BAD Bcl-2 Bcl-XL c-Jun c-Myc CDK CDKI Chk1 Cip CK2 CREB eEF eIF ERK FOXO FRP GβL

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GLUT GSK-3 HIF-1α HM HSF-1 HSP I-R

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eIF4E binding protein apoptotic protease activating factor 1 apoptotic signal-regulated kinase 1 (also called MAPKKK5) Bcl-2-associated death promoter B-cell lymphoma 2 B-cell lymphoma-extra large cellular-jun cellular-myc cyclin dependent kinase cyclin-dependent kinase inhibitor checkpoint kinase 1 CDK-interacting protein casein kinase 2 cAMP response element-binding eukaryotic elongation factor eukaryotic initiation factor extracellular signal-regulated protein kinase forkhead box O frizzled related protein G-protein β-subunit like protein (also called LST8) glucose transporter glycogen synthase kinase 3 hypoxia inducible factor 1, α subunit hydrophobic motif domain heat shock factor-1 heat shock protein ischemia-reperfusion

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IGF-1 IGF-1R IKK IL-1 INK4 IRS JNK Kip MAPK MAPKK MAPKKK MCL-1 MDM2 MK2 MPF mTOR mTORC1 NF-κB

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NFAT Nrf2

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p70-S6K PDGF PDK1 PGC-1α

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PH PI3K PIF PIP PKB PRAS40 PTEN RAC-PK Raptor Rictor ROS SAPK SGK S6K

-

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insulin-like growth factor-1 IGF-1 receptor IκB kinase interleukin-1 inhibitor of CDK4 insulin receptor substrate c-Jun N-terminal kinase kinase inhibition protein mitogen-activated protein kinase MAPK kinase MAPK kinase kinase, also known as MEKK myeloid cell leukemia sequence 1 murine double minute 2 MAPK-activated protein kinase-2 M-phase promoting factor mammalian target of rapamycin mTOR complex 1 nuclear factor kappa-light-chain-enhancer of activated B cells nuclear factor of activated T-cells NFE2L2, or nuclear factor (erythroid-derived 2)like 2) ribosomal protein S6 kinase, 70 kDa platelet-derived growth factor phosphoinositide-dependent kinase-1 peroxisome proliferator-activated receptor gamma coactivator 1-alpha pleckstrin homology domain Phosphoinositide 3-kinase PDK1-interacting fragment phosphatidylinositol phosphate protein kinase B Proline-rich Akt Substrate 40 kDa phosphatase and tensin homologue related to the A and C protein kinase regulatory associated protein of mTOR rapamycin-insensitive companion of mTOR reactive oxygen species stress-activated protein kinase Serum/glucocorticoid regulated kinase ribosomal protein S6 kinase

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SH2 domain-containing inositol 5â&#x20AC;˛-phosphatase tumor necrosis factor tuberous sclerosis wingless int

1. Introduction All living organisms rely on sequences of biological events which interpret external stimuli and trigger a network of intracellular signalling cascades that restructure the cellular environment. Signal transduction is the key event for launching the stress response. External stress signals trigger changes to the existing protein/enzyme machinery (e.g. via posttranslational modifications such as protein phosphorylation) as well as changes in gene expression (e.g. via transcription factor regulation) in order to modulate the type and/or amount of regulatory factors. Therefore, when a stress occurs, it is converted into various biological signals following cell signalling pathways and leading to both modification of the properties of existing enzymes/proteins and gene transcriptional/translational events that change the numbers and types of cell proteins. The framework for these cellular changes depends on balancing contrasting signals that break down organic matter in order to harvest energy in the form of ATP (catabolic pathways), versus those that build biomolecules utilizing energy to promote growth (anabolic pathways). Therefore, the â&#x20AC;&#x153;decisionâ&#x20AC;? to promote growth, suspend life, or initiate death signals is regulated by signal transduction pathways and the harmonization of antagonistic signals that equilibrate an organism with its environment.

Studies that evaluate the relationship between stress factors and the natural responses of organisms are extremely informative as they illustrate how signalling pathways may be altered in a context dependent manner.

Using a comparative biochemical approach that analyzes multiple systems of aerobic dormancy, our lab has documented the connections between selected signalling pathways and the biochemical adjustments that regulate the ability to enter a hypometabolic state [1-3]. Hypometabolism is quite common across phylogeny and allows organisms to suspend normal life and conserve fuel/energy by transitioning into a dormant/torpid state until stressful conditions are alleviated [4]. Hypometabolism contributes to many phenomena including hibernation in cold weather, aestivation in arid/hot environments, anaerobiosis when oxygen is limiting, anhydrobiosis under ultra-dry conditions, and others such as diapause or dauer state that allow a stress-responsive pause in the normal life cycle of a species. As such,

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entrance into a hypometabolic state can essentially rescue life by broadening the range of stressors that an organism can tangibly cope with. Through alterations of metabolic pathways which inhibit the majority of biological functions, life can be put on hold and, subsequently, continue unscathed upon reversal to the active lifestyle.

Using examples from several different animal groups, this chapter focuses on aerobic dormancies, such as aestivation and hibernation, as a strategy for dealing with extreme environments. Aestivation allows organisms to survive the dry season through adaptations which improve water retention and provide sufficient fuel reserves to survive the long summer months [2]. Hibernation allows small mammals to escape the massive costs for thermogenesis that would otherwise be needed for winter survival [5]. While these strategies each possess unique sets of adaptations, all share one common feature: metabolic rate depression. By entering into a hypometabolic state where metabolic rate may be reduced by as much as 70-99% compared with active states, animals greatly extend the time that they can survive under stressful conditions [4].

The regulatory mechanisms that ensure entrance into, long-term maintenance, and arousal from the hypometabolic state depend on innumerable regulatory circuits which converge in an intricate fashion. These mechanisms may include the complete suppression, reversal or maintenance of central metabolic pathways such as glucose metabolism, protein synthesis, cell cycle, and apoptosis [6]. The regulation of carbohydrate metabolism is the first and most direct path towards survival, providing both the building blocks and the energy for anabolic functions. Matched with signals that increase rates of protein synthesis and promote cell cycle progression, the cell can grow and proliferate in its environment. By contrast, there is constant competition from catabolic processes and the pathways which degrade cellular machinery and initiate programmed cell death. Adding yet another layer of regulation, these key pathways often demonstrate extensive crosstalk, relaying messages in order to ultimately arrive at a concerted response.

A common denominator in all of these metabolic pathways is the regulation of the serine/threonine protein kinase Akt (also known as protein kinase B, PKB) and the multitude of downstream effects associated with this pivotal stress kinase [7-8].

Since the first reports about Akt in 1991, this enzyme has been considered to be one of the most important players in cell signalling [9-11].

Akt is mainly involved in growth factor regulated pathways under the upstream control of PI3K (phosphoinositide 3-kinase) and has come to prominence because of its abundant downstream actions including controls

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on glucose metabolism, protein synthesis, cell proliferation (related to cell cycle), cell survival (related to apoptosis), etc., as well as its involvement in human disease pathologies [7-8]. Furthermore, Akt participates widely in crosstalk with other signalling pathways (e.g. those controlled by Nf-κB, ERK, Wnt), indicating a critical position of PI3K-Akt in cell signalling [12-16]. Most importantly, the PI3K-Akt signalling cascade responds robustly to stress signals [17].

Whereas an overwhelming body of research is dedicated to unravelling the regulatory secrets of cell survival and metabolism, many aspects of even the most well-defined signalling pathways remain puzzling. Therefore, the aim of this chapter is (1) to present an overview of the PI3K-Akt signalling pathway, (2) highlight the mechanisms of Akt regulation in glucose metabolism, protein synthesis, cell cycle and apoptosis, and finally (3) outline the role of Akt in the regulation of hypometabolism with examples ranging from invertebrates to vertebrates.

2. Akt and the PI3K-Akt pathway 2.1. Akt/PKB family Akt is a 56 kDa serine/threonine protein kinase that is also known as PKB or as RAC-PK (Related to the A and C Protein Kinase). It belongs to the AGC protein kinase family that includes over 60 members among them protein kinases A, G and C as well as S6K (ribosomal protein S6 kinase), SGK (Serum/glucocorticoid regulated kinase), PDK1 (phosphoinositidedependent kinase-1), etc. [18]. Members in this kinase family share a similar structure of their catalytic domain, as well as the mechanism by which they are activated. The growth factor and insulin responsive AGC kinases are upstream of a variety of biological events, including the regulation of metabolism, cell growth, proliferation, and survival [19]. Multiple upstream factors affect Akt including epidermal growth factor, insulin, IGF-1 (insulinlike growth factor-1) and PDGF (platelet-derived growth factor) [20]. In addition, Akt can be regulated in response to environmental stresses including heat shock and hypoxia, among others [20-27].

Three isoforms of Akt have been identified to date in mammalian tissues: the original Akt form (also known as Akt1, PKBα, or RAC-PKα), Akt2 (PKBβ or PAC-PKβ) and Akt3 (PKBγ or PAC-PKγ). The three isoforms are widely and differentially expressed, each tissue in mammals having at least one isoform. The expression pattern of Akt seems related to the differentiation level of cells with a higher expression level in terminally differentiated cells [28-29].

The amino acid sequences of the three isoforms have a high degree of

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similarity. Like most of the AGC kinases, all three Akt isoforms have a PH (pleckstrin homology) domain in the N terminus, a central kinase/catalytic activity domain, and a C terminal HM (hydrophobic motif) domain [18]. All three Akt isoforms follow the same pattern of regulation [28-29].

2.2. Upstream regulatory signals and Akt activation The full expression of Akt activity requires phosphorylation at multiple sites and includes translocation to the plasma membrane where the enzyme is influenced by phospholipid signalling molecules (Fig. 1). In the case of Akt1, two core sites must be phosphorylated to stimulate kinase activity: S473 in the HM domain and T308 in the catalytic domain T-loop [30]. T308 has long been known to be phosphorylated by the upstream kinase PDK1 that also phosphorylates the catalytic domain of most AGC kinases [30-31] whereas, more recently, the kinase that phosphorylates S473 was determined to be mTORC2 (mammalian Target of Rapamycin Complex 2) [32-33]. To summarize the regulation of Akt, it is easiest to start with upstream signalling events occurring at the plasma membrane.

Figure 1. Upstream regulatory signals and Akt activation. (A) Recruitment of Akt to plasma membrane by PIP2/PIP3; (B) phosphorylation of Akt by mTORC2; (C) interaction between Akt and PDK1 via the Akt PIF pocket; (D) the release of PDK1, the active Akt, and stabilization of Akt via an activation loop.

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Multiple upstream factors are involved in Akt/PKB signalling at the plasma membrane including membrane receptors, adapter proteins, class I PI3K, PTEN (phosphatase and tensin homologue), and SHIP (SH2 Domaincontaining Inositol 5′-Phosphatase). PI3K is the main direct factor involved in Akt regulation and addition of PI3K inhibitors prevents phosphorylation of Akt [34]. PI3K can be activated by either receptor tyrosine kinases (Class IA), such as IGF-1R (IGF-1 receptor), or G-protein coupled receptors (Class IB), such as members of the Ras superfamily of small GTPases [35]. For example, IGF-1 binds specifically to the receptor tyrosine kinase IGF-1R which, following receptor autophosphorylation and activation of receptor tyrosine kinases, phosphorylates and activates PI3K (Fig. 1A). [24].

At the plasma membrane, PI3K produces the phosphatidylinositol-3,4,5trisphosphate (PI[3,4,5]P3 or PIP3) from phosphatidylinositol-3,4-bisphosphate (PI[3,4]P2 or PIP2) [35]. This reaction is opposed by at least two different phosphatases; the phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase PTEN and the inositol 5-phosphatase SHIP [36]. PTEN dephosphorylates the 3-position of the inositol ring to convert PIP3 to PI[4, 5]P2 [37] and the inhibition of PI3K under conditions where PTEN was activated caused a rapid drop in PIP3 levels [38]. By contrast, SHIP specifically recognizes and removes phosphate at the 5-position on PIP3 to produce PI[3, 4]P2 [36] and SHIP-null mice show increased PIP3 levels [39]. Although signalling downstream of PI3K is weakened through the dephosphorylation of PIP3 by SHIP, PI[3, 4]P2 may also activate downstream responses which are both PI3K-dependent and PIP3-independent [36, 40-41].

Both PIP3 and PI[3,4]P2 are responsible for recruitment of Akt and PDK1 to the plasma membrane through interactions with their PH domains (Fig.1A) [40-43]. Andjelkovic et al. [44] showed that after treatment with IGF-1, native Akt was almost immediately localized to the plasma membrane whereas a mutant Akt with a deleted PH domain did not translocate.

However, Akt1 constructed without the PH domain could still be activated and/or phosphorylated even though no plasma membrane translocation was detected [44]. Studies also showed that the PH domain-dependent interaction between Akt and PIP3 failed to stimulate Akt activity in vitro, and treatment with PI[3,4]P2 could not activate Akt catalytic activity dramatically in vitro [40-41]. Therefore, it is hypothesized that the PH domain does not directly stimulate catalytic activity, but rather creates a greater chance for interaction of the PDK1 catalytic domain with Akt at the plasma membrane.

As mentioned above, mTORC2 is now known to be the kinase that phosphorylates Akt/PKB at S473 (S474 in Akt2; S472 in Akt3) [32-33]. This is a large multi-subunit complex consisting of mTOR, Rictor (Rapamycininsensitive companion of mTOR) and GβL (G-protein β-subunit like protein)

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(Fig. 1B). In contrast to mTORC1 (mTOR complex 1) (discussed below under protein synthesis regulation), mTORC2 is not rapamycin sensitive and its cellular functions are only beginning to be discovered [33]. Whereas S473 phosphorylation plays a regulatory role in the activation event, it has little effect on kinase activity when presented alone [45]. Similarly, other phosphorylation events in the HM domain such as Y474 were also reported to contribute to the conformational change occurring during Akt activation [46].

Following S473 phosphorylation, Akt1 is next phosphorylated on T308 (T309 in Akt2; T305 in Akt3) by PDK1 which also phosphorylates the catalytic domain of most AGC kinases (Fig. 1C) [30-31]. PDK1 is considered to be a special AGC kinase that contains a PH domain and catalytic domain, but lacks a HM domain [31]. Due to the role of the PH domain in recruitment to the plasma membrane, PDK1 is only able to activate Akt in a PIP3/PI[3,4]P2-dependent manner [40] and deletion of the PH domain attenuates PDK1-dependent Akt activation [47-48]. In addition, PDK1 interacts with Akt partially through the PIF (PDK1-interacting fragment) pocket in the Akt HM domain as also occurs for the PDK1-dependent phosphorylation of other AGC kinases [31, 49]. One possible explanation for this interaction is that the PIF pocket with phospho-S473 provides PDK1 with a docking site to create a physical interaction for exerting its kinase activity on Akt [31, 49].

Finally, following phosphorylation by PDK1 at T308, Akt1 is in a stabilized conformation and is free to move away from the plasma membrane (Fig. 1D). Once free, several additional phosphorylation sites have been reported to play a role in Akt activation. For example, tyrosine phosphorylations at Y315 and Y326 within the catalytic domain appear to occur after activation by Akt upstream signals and mutations on these two sites hindered Akt activation [50]. Finally, Akt achieves a stabilized conformation and is primed to initiate interactions with a multitude of downstream targets.

3. Downstream effects of Akt signaling Akt acts on a variety of proteins and phosphorylates Ser/Thr residues that occur in a characteristic sequence, Arg-X-Arg-X-X-(Ser/Thr), on the substrate protein [51]. Yang et al. (2002) identified the structural basis for Akt activation and the substrate motif via analysis of the crystal structure of purified Akt. Proteins presenting this structural motif can be considered as potential Akt targets. Therefore, a wide range of putative targets downstream of PI3K-Akt signalling have been revealed [52]. They are involved in regulating various vital biological processes (Fig. 2).

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Figure 2. The involvement of Akt in regulating various vital biological processes including: (A) glucose metabolism, (B) protein synthesis, (C) cell cycle, and (D) apoptosis. Akt exerts its regulatory effects via the phosphorylation of downstream proteins. Akt regulates glucose metabolism through GSK-3, CREB, NFAT, and GLUT1/4; protein synthesis through TSC2 and PRAS40; cell cycle through p27Kip1, FOXO, MDM2, Wee1 and Myt1, and ChK1; apoptosis through Bad, Caspase-9, and FOXO. Arrowheads denote positive regulatory effects while blunt-ended lines denote negative regulatory effects. An "X" denotes the action is inhibited.

3.1. Regulation of glucose metabolism PI3K-Akt signalling exerts control over glucose metabolism in two central ways; (1) release of the inhibitory effects of GSK-3 (glycogen synthase kinase 3), and (2) altered expression levels of glucose transporters [53-54]. GSK-3 is a serine/threonine protein kinase encoded by two isoforms in mammals, GSK-3α and GSK-3β, and has been implicated in a wide range of cellular functions (e.g. metabolism, differentiation, cell fate determination) [6, 53, 55]. In general, GSK-3 phosphorylation has an inhibitory effect on its downstream targets thereby causing them to be recognized and degraded by ubiquitin proteosomal pathways [56]. These inhibitory effects change the ability of substrate enzymes to catalyze chemical reactions or substrate transcription factors to localize to the nucleus to initiate gene transcription. Akt lies upstream of GSK-3 and phosphorylates S21 on GSK-3α or S9 on GSK-3β, thus inactivating GSK-3 and preventing its inhibitory downstream action (Fig. 2A) [53].

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GSK-3 is so named because glycogen synthase was the first downstream target of the kinase to be recognized [55]. GSK-3 inactivates glycogen synthase by the sequential phosphorylation of residues S652, S648, S644 and S640 and this blocks glycogen synthesis, particularly at times when glycogen catabolism is required [57]. GSK-3 also inhibits various transcription factors including CREB (cAMP response element-binding), c-Jun (cellular-Jun), c-Myc (cellular-Myc), HSF-1 (heat shock factor-1), NFAT (nuclear factor of activated T-cells), and GATA-4 [53, 58-59]. In addition, GSK-3 regulates the Wnt (wingless int) signalling pathway (see Cross-talk section).

Akt promotes glycolysis through its positive effect on glucose transporters (GLUT) [54, 60-61]. Akt up-regulates the expression of GLUT1 (responsible for basal glucose uptake in cells) through the activation of the mTORC1 pathway (see Protein synthesis regulation) [62]. Activation of mTORC1 promotes the expression of the transcription factor HIF-1α (Hypoxia Inducible Factor-1α), which lies upstream of GLUT1 [63]. In addition, the expression level of GLUT1 can be up-regulated through a CAPdependent translational activation (mediated by eIF4E) which relies on the inhibition of 4EBP (eIF4E binding protein) by mTORC1 [62]. Akt also plays a role in the regulation of GLUT4 (the insulin-regulated transporter) by promoting its translocation to the plasma membrane [26]. Moreover, it has been reported that the Akt–dependent inhibition/phosphorylation of both FOXO1 (see Apoptosis regulation) and PGC-1α (peroxisome proliferatoractivated receptor gamma coactivator-1α) [65] has the potential to modulate hepatic glucose levels [64].

3.2. Regulation of protein synthesis A central action of PI3K-Akt signalling is the regulation of protein synthesis through the control of the mTOR (mammalian target of rapamycin) pathway. mTORC1 is formed with mTOR, Raptor (Regulatory associated protein of mTOR) and GβL (G-protein β-subunit like protein, also called LST8) (Fig. 2B). One of the main inhibitors of mTORC1 is the Tuberous sclerosis complex (TSC1/TSC2) which exerts its control through RHEB. The inhibitory mechanism depends on the GTPase activity of TSC2 which converts the GTP-bound, active RHEB (Ras homologue enriched in brain) into the GDP-bound, inactive form [66]. The PI3K-Akt pathway plays an upstream regulatory role by inhibiting TSC2 activity and therefore indirectly promoting mTORC1 activity. TSC2 can be phosphorylated by active Akt on three sites (S939, S981 and T1462) [67-69] where it is then recognized by 14-3-3 and prevented from forming a complex with TSC1 [70].

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A newly discovered protein, PRAS40 (Proline-rich Akt Substrate 40 kDa), is believed to be another negative regulator of mTORC1 [71-72]. PRAS40 inhibits the kinase activity of mTORC1 by a physical interaction with Raptor [73]. As indicated by its name, PRAS40 is a major target protein of Akt. Active Akt phosphorylates PRAS40 at T246 to trigger a 14-3-3 protein interaction which sequesters PRAS40 from binding to mTORC1 [71].

Active mTORC1 promotes protein synthesis by phosphorylating targets including 4EBP and p70-S6K (ribosomal protein S6 kinase, 70 kDa).4EBP is a negative regulator of ribosomal eIF4E (eukaryotic Initiation Factor 4E).

The hypo-phosphorylated form of 4EBP binds to eIF4E to prevent it from interacting with 5’ cap-containing mRNAs to form the pre-initiation complex [69, 72-73]. Active mTORC1 hyper-phosphorylates 4EBP at multiple sites, thereby blocking its inhibitory interaction with eIF4E and allowing mRNA transcripts to be brought into assembling ribosomes. For the AGC kinase p70-S6K, on the other hand, phosphorylation on T389 by mTORC1 is a prerequisite for its PDK1-dependent full activation [74-75]. Active p70-S6K then phosphorylates eIF4B which in turn contributes to the helicase activity of eIF4A to promote mRNA translation [76-78].

Hence, in general, the PI3K-Akt pathway acts as an activator of mTORC1, and thereby promotes protein synthesis. However, a negative feedback loop regulating the process has also been proposed. After stimulation by insulin, the activated insulin receptor phosphorylates IRS (insulin receptor substrate) at a tyrosine site, and the phospho-IRS then activates PI3K to produce PIP3 and P[3, 4]IP2 which leads to Akt activation.

Activated Akt then stimulates mTORC1 which phosphorylates p70-S6K. Subsequently, activated p70-S6K phosphorylates IRS at a serine site to promote its degradation and thereby terminates the signalling cascade [79-81]. In addition, it has been reported that PTEN can also regulate Akt in the negative feedback loop, albeit in an indirect fashion [82].

PI3K-Akt signalling may also promote protein synthesis in an mTORC1independent mechanism. GSK3β can also phosphorylate eIF2B (eukaryotic initiation factor 2B) at S540, causing inhibition of its guanine nucleotide exchange activity and preventing it from recharging the GTP residue on eIF2α [83]. Insulin treatment released this inhibition via inactivation of GSK3β [83]. Therefore, Akt-dependent inhibition of GSK3β may be involved as an upstream factor in this event.

3.3. Cell cycle control Another aspect of PI3K-Akt action is the regulation of cell cycle progression [84]. The cell cycle has four main phases: G1 (growth), S (DNA

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replication), G2 (preparation for mitosis), and M (mitosis). The cell cycle can be regulated by various factors and events in each phase, the crucial regulatory events being mediated by a group of dimeric proteins that each consist of one cyclin dependent kinase (CDK) (the catalytic subunit) and one cyclin (the regulatory subunit). The two most important regulatory periods are the G1/S phase transition and the G2/M phase transition. For the G1/S transition in eukaryotic cells, CDK2, -4, and -6 along with cyclins A, -D1, -D2, -D3 and -E play crucial roles, whereas CDK1 (also known as Cdc2) and cyclins A, -B1, -B2 are vital to the G2/M phase transition. The CDK1/Cyclin A (-B) complex is also called MPF (M-phase promoting factor), with the function of driving the cell cycle into M phase [85].

The activity of most regulatory CDK/cyclin complexes is related to their phosphorylation state. For example, for the G2/M phase transition, a set of protein kinases are of great importance including Cdc25, Wee1, and Myt1 [86] and these are opposed by CDKIs (CDK inhibitors). Comparable effectors that act at the G1/S phase transition include members of the INK4 (inhibitors of CDK4) family (p16INK4A, p15INK4B, p18INK4C, p19INK4D) that are specific to CDK 4, 6; and the Cip/Kip (CDK-interacting protein/Kinase inhibition protein) family (p21Cip1, p27Kip1, p57Kip2) that regulate CDK2: Cylin E1/E2 [87]. The family of 14-3-3 proteins is also key in cell cycle control. Various cell cycle regulatory factors can be sequestered away from the nucleus by 14-3-3 proteins in response to different signals, thereby limiting their function [85].

The positive regulatory effects of the PI3K-Akt cascade on the cell cycle come largely from the inhibition of CDKIs (Fig. 2C). For example, phosphorylation of p27Kip1 at T157 by Akt contributes to its sequestration by 14-3-3 protein, which prevents p27Kip1 from localizing into the nucleus. In addition, Akt inhibits the specific FOXO transcription factor that regulates p27Kip1 to suppress expression of this protein [88]. Another CDKI, p21Cip1, is also inhibited by Akt in an indirect fashion: Akt activates MDM2 (murine double minute 2) by phosphorylation which in turn promotes the degradation of the transcription factor p53 that controls p21Cip1 expression [89-92]. The phosphorylation of MDM2 by Akt at sites of S166 and S188 specifically stabilizes MDM2 and inhibits its self-ubiquitination, thus preventing it from degradation by proteases [93].

Akt also has negative effects on G2/M phase transition regulatory kinases, including Wee1 and Myt1 [94-96], both of which contribute to inhibitory phosphorylation of CDK1. Inactivation of Wee1 and Myt1 by Akt phosphorylation ensures that CDK1 remains in its activated, nonphosphorylated form. In addition, direct phosphorylation of the DNA damage Chk1 (checkpoint kinase 1) by Akt at S280 blocks its translocation into the

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nucleus, thereby preventing Chk1 from phosphorylating Cdc25. Thus, Cdc25 is activated and via its phosphatase activity may dephosphorylate/activate p-CDK1 and allow cells to enter M phase [97]. As mentioned above, the PI3KAkt pathway activates mRNA translation by the Akt-TSC-mTOR-4EBP pathway and this activation promotes the translation of many genes, including cyclin D1, which is required for cell cycle progression [98]. Furthermore, the first identified PI3K-Akt pathway substrate, GSK3, is also an important negative regulator of Cyclin D and E (via protease degradation) [99-100].

3.4. Apoptosis regulation The starting event in apoptosis is the release of cytochrome C from mitochondria into the cytoplasm under the control of the antiapoptotic Bcl family member, Bcl2. Cytochrome C forms a so-called apoptosome in conjunction with the adapter protein Apaf-1 (Apoptotic protease activating factor 1), caspase 9 and dATP. This interaction activates caspase 9 which in turn initiates the upcoming proteolysis by activating other caspase members, including caspase 3 and 7 [101-102]. PI3K-Akt signalling contributes to the regulation of programmed cell death by controlling several key factors including BAD (Bcl-2-associated death promoter), caspase 9, GSK3β, and the FOXO family of transcription factors. In general, the PI3K-Akt pathway has a prosurvival role and is active when nutrients and energy are plentiful; hence, Akt action typically inhibits apoptosis (Fig 2D).

Unphosphorylated BAD can bind and inhibit the prosurvival Bcl-2 family protein, Bcl-XL (B-cell lymphoma-extra large), which interacts with Apaf-1 to prevent the activation of caspase 9 and thereby suppresses apoptosis [103]. Akt exerts inhibitory effects on the proapoptotic protein BAD by phosphorylating the protein at S136 and facilitating a subsequent 14-3-3 protein-dependent sequestration event [104-105]. Evidence of the importance of Akt-mediated BAD phosphorylation, is demonstrated by increased apoptosis rates when a wild type BAD is replaced with a mutant at S136 [104].

Furthermore, caspase 9 can be directly phosphorylated by Akt at S196 [106] resulting in a decrease in its proteolytic activity. Indeed, when S196 was replaced by A196, caspase 9 showed greater proapoptotic activity than the wild type in the presence of active Akt. In addition to regulating factors that are directly involved in the apoptosis process, Akt also controls the FOXO transcription factors that contribute to the expression of apoptosis-requiring components. This includes up-regulation of the death signal factor cytokine Fas ligand. The Fas ligand then binds to the Fas receptor on the cell surface to induce apoptosis by activating caspases in an indirect manner. Akt directly phosphorylates

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FOXO1, FOXO3a and FOXO4 in the nucleus, each at three sites. These phospho-FOXOs can then be sequestered by 14-3-3 proteins in a similar manner to the Akt-dependent regulation of BAD and other targets mentioned above [reviewed in 107]. Therefore, Akt inhibits FOXO transactivation activity by preventing it from localizing into the nucleus and, thereby, indirectly suppresses apoptosis.

Akt may also control apoptosis via GSK3β. The prosurvival factor, MCL-1 (myeloid cell leukemia sequence 1), of the Bcl-2 family is reported to be directly inhibited by GSK3β [108]. Therefore, an inhibition of GSK3β by Akt has potential effects on the GSK3β-dependent inhibition of MCL-1.

Crosstalk between PI3K-Akt and other signalling pathways (NF-kB, JNK/p38 pathway, etc.) also provides some lines of evidence for Akt-dependent promotion of cell survival (see Crosstalk section).

4. Akt and crosstalk with other signalling pathways In addition to the direct effects of PI3K-Akt signalling described above, the pathway also interacts with other major signalling pathways. This crosstalk extends its regulatory importance in terms of the stress response, especially the interactions with the NF-κB, Wnt, and MAPK pathways.

4.1. Akt and NF-κB The NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) signalling pathway regulates functions including innate immunity, cell proliferation, and apoptosis, among others [109-111]. A wide range of signal proteins including TNF (tumor necrosis factor), IL-1 (interleukin-1), and upstream signalling pathways such as JNK/p38 signalling are able to activate the NF-κB pathway [112-114].

NF-κB features five members sharing a Rel homology domain: p65 Rel A, C-Rel, Rel B, p52 and p50. The first three have a C-terminal transactivation domain, whereas the other two (p52 and p50) only have an inhibitor binding region (ankyrin repeats) shared by the inhibitory factor IκB [115]. Because they lack the transactivation domain, p52 and p50 do not have transactivation activity unless they form dimers with p65 RelA, RelB or C-Rel. In most cases, the active NF-κB is a heterodimer composed of p65 and p50, but another common form of active NF-κB is the p52/Rel B dimer [115]. Activation of NF-κB by its upstream pathways all lead to the formation of the NF-κB heterodimer, either via protease degradation of the inhibitory factor IκB or the processing of the regulatory heterodimer subunit p105 to make the active p50 that forms a dimer with p65. The ubiquitination-dependent protease degradation of IκB

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requires phosphorylation. The upstream kinases for such inhibitory phosphorylation include IKKs (IκB kinase) and CK2 (Casein kinase 2) which is regulated through JNK/p38 signalling [115]. IKKs also require a phosphorylation-dependent activation to exert their catalytic activity on IκB [115]. It has been reported that under PDGF stimulation, TNF can induce the activity of PI3K which in turn activates Akt, eventually leading to an increase in NF-κB DNA binding activity [12]. Also, Akt can phosphorylate/activate IKKα at T23, triggering IKK-dependent NF-κB activation [116-117].

4.2. Akt and WNT signalling through GSK3β Although originally identified for its involvement in embryogenesis, the Wnt pathway is now known to be involved in regulating many different biological events [118]. The Akt downstream target, GSK3β, plays a key role in Wnt signalling. The first signal protein that was identified in this pathway was the glycoprotein Wnt which is recognized by a receptor complex composed of seven transmembrane frizzled proteins and FRPs (frizzled related proteins). However, besides Wnt, it has been reported that the Wnt signalling pathway can also be activated by multiple other signals, such as insulin, IGF-1, PDGF, cAMP, and protein kinase A [119]. β-Catenin is the core functional component of the pathway which goes into the nucleus and contributes to target gene expression when activated. Otherwise, β-catenin is tightly held by a so-called “destruction” complex containing GSK3β, APC and Axin. GSK3β phosphorylates and destabilizes β−catenin at three sites (S33, S37, T41), leading to subsequent protease degradation [120]. When the pathway is stimulated, β-catenin is released from the “destruction” complex and translocates into the nucleus. The nuclear β-catenin interacts with the co-factor TCF4 to become a dual transcription factor.

Also, β-catenin can act as a co-factor of the transcription factor FOXO contributing to its transactivation activity [121].

As mentioned above, the phosphorylation of GSK3β at S9 by Akt attenuates its catalytic activity. Therefore, Akt may contribute to Wnt signalling activation, although studies have shown contrary results in different cell types [16, 122-123]. It has been reported that in intestinal cells, the translation level of Cyclin D1 and c-Myc genes downstream to Wnt signalling were stimulated in an Akt/mTOR-sensitive manner [16]. This would suggest a potential regulatory role of PI3K-Akt on Wnt signalling activation. Furthermore, the well-defined Akt downstream target, mTOR, can be effectively activated by Wnt signalling via GSK3β [124]. According to these findings, it appears PI3K-Akt signalling and its downstream targets have the potential to activate the Wnt pathway and vice versa.

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4.3. Akt and MAPK pathways In addition to NF-ÎşB and Wnt signalling, Akt also interacts with MAPK (mitogen-activated protein kinase) signalling pathways, including the JNK/SAPK (c-Jun N-terminal kinases or stress-activated protein kinases), p38 family kinases, and ERKs (extracellular signal-regulated protein kinases) [125].

MAPKs are serine/threonine protein kinases that respond to a wide range of extracellular stimuli (for example, growth factors, cytokines, stress) and have been identified as regulators of many cellular activities (e.g. oxidative and osmotic stress, apoptosis, mitosis, cell differentiation and proliferation, immune response, memory, etc.) [1]. A comprehensive description of MAPK regulation is beyond the scope of the present article but the reader is directed to Cowan & Storey (2003) for more information. Briefly, all MAPK related pathways follow a common pattern of activation which begins with an extracellular signal that interacts with cell surface receptors, GTP-binding proteins or with other kinases [1]. These messages are transmitted through the cytosol to the nucleus by the sequential phosphorylation of MAPKKK (MAPK kinase kinases), MAPKK (MAPK kinases), and MAPKs. The MAPKs then phosphorylate their target proteins (e.g. transcription factors) which alter gene expression and modulate the cellular environment.

The stress-responsive JNK/p38 pathway can be inhibited by Akt via phosphorylation of the JNK/p38 upstream factor ASK1 (Apoptotic signalregulated kinase 1, or MAPKKK5) at S83, which prevents ASK1 from being activated by apoptotic stimuli [14]. In addition, p38 regulates the activation of Akt through a complex with other factors including HSP27 (heat shock protein 27) and MK2 (MAPK-activated protein kinase-2) in human neutrophils [15, 126]. Therefore, Akt can promote cell survival through both inhibitory effects on proapoptotic factors and the apoptotic signalling pathways. Akt, as an oncoprotein, inhibits the ERK pathway by directly phosphorylating c-Raf at T259 in a human breast cancer cell line, thereby bypassing the normal cell cycle arrest function of ERK signaling [13].

Another study from the same group showed that the interaction between the PI3K-Akt pathway and ERK signalling only occurred in certain stages of muscle development [127].

5. Hypometabolism and survival in extreme environments All organisms must deal with variation in their environment involving a wide range of factors, both physical and biological, and occurring on time scales from minutes to years. As a result, organisms require adaptive strategies that allow them to take advantage of favourable conditions (and

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quickly promote growth and development) and alleviate stresses associated with unfavourable ones. One of the more dramatic ways of dealing with severe stress is to conserve energy reserves by strongly suppressing metabolic rate and entering a hypometabolic state. Hypometabolism (also known as dormancy, torpor) characterizes a wide variety of survival strategies among animals including hibernation, aestivation, anaerobiosis, anhydrobiosis, diapause, and dauer state, among others [4]. Studies that have analyzed the intracellular signalling pathways involved in mediating oscillations in stress-responsive metabolic rate have been pivotal in uncovering Akt regulatory mechanisms as well as identifying similarities in these pathways, conserved from nematodes to mammals [6, 128-129]. For example, the nematode, Caenorhabditis elegans, enters a resting or dauer state between the second and third stage of larval development and the regulation of the dauer state has been studied extensively [6]. In the dauer condition, a dramatic suppression of metabolic activity produces energy savings that can sustain the nematode through periods of limited food, fluctuating temperatures, and overcrowding. Many insects also exhibit a period of dormancy, or diapause, during at least one stage of their life cycle whether it be during egg, pupal, larval or adult life stages [128]. This includes model organisms such as the common fruit fly, Drosophila melanogaster, and the silkworm, Bombyx mori, and in both cases, links have been made to Akt regulation [128].

The Akt pathway in C. elegans and D. melanogaster is regulated by insulin receptors, the phosphorylation of PI[3, 4]P2 by PI3K, the dephosphorylation of PIP3 by PTEN, and recruitment of PDK and Akt to the cell membrane [6, 128]. In addition, the downstream effects of Akt activation parallel those seen in mammalian model organisms exerting control over glycogen biosynthesis, protein synthesis, cell cycle, and apoptosis [6, 128]. Studies with nematodes and fruit flies have shed light on multiple facets of PI3K-Akt signalling and several review articles have already described the activation, regulation, and downstream effects of Akt networks during dauer and diapause states [130-133]. Thus, the following sections focus on other forms of aerobic dormancies, such as aestivation and hibernation, not yet highlighted in the literature.

Even though individual species may experience unique environmental challenges, all strategies that deal with extreme environments contain parallels in the mechanisms by which hypometabolism is achieved. For example, the controlled suppression of metabolic rate is matched with adaptations which reprioritize ATP use by many different cellular processes.

This cellular reorganization relies on the concerted effects of signal transduction pathways that adjust the amount/type of biomolecules in order to

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support hypometabolism. Therefore, by comparing and contrasting Akt related signalling pathways and the stress response over a range of stressresponsive strategies, we will unravel the regulatory mechanisms of this central metabolic pathway and potentially shed light on its involvement in hundreds of disease pathologies. The following sections begin with a brief description of some of the common forms of aerobic dormancies and, subsequently, outline the secrets uncovered about Akt regulation in nature.

5.1. Aestivation Aestivation is a state of aerobic torpor which is triggered primarily by arid conditions (low water stress) but is also often associated with high temperatures and low food availability [2]. Suppression of physiological functions such as breathing and heart rate as well as reduced muscle activity contribute to energy savings in the hypometabolic state [134]. In addition, multiple biochemical adjustments mediated by mechanisms such as reversible protein phosphorylation, manipulation of signal transduction networks, and differential gene expression, ensure that aestivators are able to achieve both coordinated entry/exit from the torpid state and also sustain long term viability while hypometabolic [2]. Although utilized by many different species, research on the molecular mechanisms of aestivation has focused mainly on a few model species including various pulmonate land snails (Otala lactea, Helix aspersa and others), lungfish, and desert toads and frogs [2,134].

As a first line of defense, aestivators seek refuge in locations adequately sheltered from the external environment [136]. In addition, mechanisms that minimize water loss are key and include the acquisition of large body water stores prior to aestivation and a switch to apnoic breathing patterns that minimize water loss during breathing [137]. Evaporation of water from the body can also be suppressed by either a physical barrier (e.g. snails secrete a mucus epiphragm over the opening of the shell) [138] or biochemical means (e.g. extreme elevation of solutes such as urea to retard water loss in a colligative manner) [134]. Nonetheless, aestivators do lose substantial amounts of body fluids over what can be 9-10 months of dormancy and, thus, they typically also exhibit improved tolerances for tissue desiccation. For example, spadefoot toads (Scaphiopus couchii) readily survive the loss of as much as 60% of their total body water [134].

5.2. Mammalian hibernation Hibernation allows many small mammals to survive deep cold and limited food availability over the winter months. By strongly suppressing

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metabolic rate, often to <5% of normal resting rate, and letting body temperature fall to ambient, many small mammals can achieve energy savings of about 90% compared with the costs of remaining euthermic over the winter months [139,140]. The hibernation season consists of long bouts deep cold torpor (days to weeks) interspersed with brief arousals back to euthermia (usually < 24 hours) [141]. While torpid, animals experience greatly reduced organ perfusion rate (<10%), heart rate (from 350-400 to 5-10 beats/min), and breathing rate (from >40 to <1 breath/min) as well as a generalized suppression of all other physiological functions [142]. Between hibernation bouts, animals rewarm themselves using heat produced from nonshivering thermogenesis in brown adipose tissue and shivering thermogenesis by skeletal muscle [3]. Interbout arousals are the most energetically costly part of hibernation and while their purpose is not fully understood, they may be important for several reasons including (1) refresh the immune system, (2) restore neural circuits, and (3) renew essential biomolecules [142].

6. Stress response by PI3K-Akt during hypometabolism 6.1. Evidence of PI3K-Akt regulation during aerobic dormancy A recent study has shown that aestivating land snails, Otala lactea, show unique PI3K-Akt signalling patterns [143]. A significant increase in total Akt protein and in phospho-Akt (S473) content occurred in both foot muscle and hepatopancreas in aestivating compared with active snails (Fig. 3). Kinetic assays also showed that the activity of Akt increased in both tissues, as well as affinity for a model peptide substrate. Several downstream targets of Akt were also investigated in the same study. The amount of phospho-FOXO3a (S253) and phospho-BAD (S136) rose significantly in both tissues during aestivation (total protein did not change) (Fig. 4 A) whereas mTOR showed no change in either S2481 or S2448 phospho-forms. Activity of GSK3 (both α and β isoforms) increased in both tissues during aestivation but total GSK3β protein did not change and the amount of phosphorylated GSK3β (S9) decreased significantly (Fig. 4 B).

The results for FOXO3a and BAD correlated well with the observed activation of Akt. During aestivation, increased levels of phospho-Akt as well as the subsequent inactivation of FOXO3a and BAD by Akt phosphorylation, suppresses pathways that induce apoptosis and, thus, contribute to cellular survival in the hypometabolic state. Despite the involvement of Akt in FOXO3a and BAD regulation, the activity and expression levels of the growth and metabolism related factors (mTOR and GSK3β, respectively) did not show consistent connections with the upstream Akt data. This suggests

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Figure 3. Protein levels and phosphorylation state of Akt in foot muscle and hepatopancreas of land snails Otala lactea. Top: Representative bands at 60 kDa from three independent samples in each condition showing Akt and phospho-Akt level.

Bottom: Histogram shows the ratio of normalized mean band intensity (Âą s.e.m., n=3) for aestivated versus active snails. a â&#x20AC;&#x201C; values for aestivation significantly different from active snails (P <0.05) [From 143].

that different downstream targets of PI3K-Akt signalling may be selectively regulated during aestivation. It may be hypothesized that mTOR was not phosphorylated and activated because the energetic costs associated with high rates of protein synthesis are not conducive to conserving limited ATP resources during hypometabolism. Indeed, additional studies in the foot muscle and hepatopancreas of O. lactea confirmed that rates of protein synthesis are dramatically reduced by about 80% during aestivation, the phosphorylation of eIF2Îą and eEF2 are significantly increased, and 4EBP1 demonstrated decreased phosphorylation levels [135]. Similarly, the inhibitory action of GSK3 on anabolic processes, such as glycogen synthesis, as well as protein synthesis, through eIF2B, results in energy savings which promote the hypometabolic state.

Another study examined PI3K-Akt in an anoxia-tolerant marine snail, the common periwinkle Littorina littorea. As opposed to the situation of aerobic hypometabolism in the land snail, entry into a state of anoxic hypometabolism in the marine snail had no effect on total Akt protein or phospho-Akt (S473) content [144]. These results emphasize a stressdependent pattern of Akt regulation during hypometabolism. It is possible that the periwinkle may utilize other intracellular mechanisms to promote survival

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Figure 4. The protein and phosphorylation levels of selected Akt downstream targets in foot muscle and hepatopancreas of land snails, Otala lactea. (A) Levels of FOXO3a and BAD. Top: Representative bands showing total and phosphorylated levels of FOXO3a and BAD in foot muscle and hepatopancreas of active and 10 days aestivated O. lactea. FOXO3a was detected as the bands at 110 kDa and BAD at 20 kDa; phospho-antibodies detected p-FOXO3a (S253) and BAD (S136), Bottom: Histograms showing normalized mean EST:ACT ratios (± s.e.m., n=5). (B) GSK-3 protein levels in foot muscle and hepatopancreas of active and 10 days aestivated O. lactea. Top: Representative bands showing total protein levels of both GSK3 subunits (GSK-3β and GSK-3α) and the levels of phospho-GSK-3β (S9). Bottom: Histograms showing normalized mean EST:ACT ratios (± s.e.m, n=5). a – values for aestivation are significantly different from active snails (P <0.05) [From 143].

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and/or limit cellular damage under anoxia such as heightened antioxidant defenses or upregulation of stress-related heat shock proteins.

Akt is also proving to be important in mammalian hibernation with a role for Akt signalling identified in two distinct hibernator groups, bats and rodents. Data from two different species of hibernating bats, Myotis lucifugus and Murina leucogaster, revealed specific expression and activation patterns of Akt [145, 146]. The findings in both species were comparable to an earlier study carried out with the greater horseshoe bat, Rhinolophus ferrumequinum [147]. Studies with little brown bats, M. lucifugus, examined the relative expression level of Akt and p-Akt (S473) in seven tissues via immunoblotting

Figure 5. Total and phosphorylated levels of Akt in little brown bats, Myotis lucifugus.

Western blotting was used to analyze total Akt protein and phospho-Akt levels (S473) in aroused (A) vs torpid (T) bats. Top: Representative bands (60 kDa) show total Akt protein levels (left) and phosphorylated Akt levels (right). Bottom: Histograms show normalized band intensities, mean Âą s.e.m., n=3. * -Values for torpid bats are significantly different from aroused animals (P <0.05) [From 145].

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[145]. Differential expression patterns were identified in selected tissues during cold torpor as compared with aroused animals (i.e. euthermic controls). In brain, heart, kidney, liver, and skeletal muscle, no significant differences in total Akt were seen during the torpid state, whereas white adipose tissue showed a significant reduction in total Akt. Heart and skeletal muscle registered no change in active Akt (phospho-S473) but the amount of p-Akt decreased significantly in brain, kidney, liver, and white adipose tissue.

However, brown adipose tissue showed a unique response: total Akt protein was enhanced as well as the amount of the active form (phospho-S473) during hibernation (Fig. 5). In general, the data agreed with the general metabolic pattern in hibernating species whereby an overall decrease in Akt activity is consistent with reduced insulin signalling during torpor [5, 145].

However, brown adipose tissue showed an unexpected up-regulation of Akt requiring further investigation. Similar to the response seen in the land snail, Akt may selectively regulate downstream signalling cascades suggesting that only certain branches of the Akt network are activated in brown adipose tissue of bats.

Studies of the pectoral muscle of the greater tube-nosed bat (M. leucogaster) delineated the role of Akt-mTOR/FOXO1 signalling in muscle mass retention during hibernation [146]. The researchers hypothesized that intermittent periods of arousal are a mechanism whereby hibernators may replenish muscle proteins depleted during hibernation.

Therefore, arousal may be viewed as a period of rejuvenation which contributes to reducing the deleterious effects of extended periods of disuse.

The ratio of p-Akt/Akt, p-mTOR/mTOR, and p-FOXO1/FOXO1 were measured in summer active versus hibernating bats and also assessed over a time course of torpor (1-7 days) and arousal. Levels of total Akt and mTOR did not change between summer active and hibernating conditions whereas p-Akt and p-mTOR levels decreased significantly. In addition, the p-Akt/Akt ratio did not vary significantly over the time course whereas the p-mTOR/mTOR ratio increased slightly after 1 day spent in torpor, decreased after 7 days in deep torpor, and returned to control levels during arousal. The overall levels of FOXO1 matched Akt expression, demonstrating no change when comparing summer versus winter animals or over the time course of torpor and arousal. The oscillation in p-mTOR expression levels indicates the possibility that protein synthesis may briefly be activated at different points over torpor-arousal in order to help maintain muscle mass. However, the activated p-mTOR expression pattern does not match the activation profile of Akt which remains unchanged throughout the time course. Further investigation is required; however, it is possible that while Akt phosphorylation levels have decreased, the enzymatic activity of the kinase

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matches the activation profile of p-mTOR. Nonetheless, it may be concluded that whereas Akt-mTOR regulation may play a role in maintaining muscle mass during hibernation, other pathways may also be involved.

In another recent study, total protein levels, enzymatic activity, kinetic properties, structural stability, and differential isozyme regulation of Akt were evaluated in Richardsonâ&#x20AC;&#x2122;s ground squirrels, Spermophilus richardsonii [129]. Immunoblotting data showed no significant change in total Akt in muscle and liver but the amount of active, phospho-S473 Akt decreased significantly in both tissues during torpor (Fig. 6). In addition, the activity of Akt, measured with a specific substrate peptide, was reduced by 60-66% during torpor as compared to euthermic controls in muscle and liver. The role of reversible phosphorylation in changing the activity state of Akt was also confirmed with in vitro incubation studies. Akt from the euthermic muscle tissue showed a dramatic decrease in activity after treatment of alkaline phosphatase, which showed that euthermic Akt was the high phosphate active form and indicated that the activity change during hibernation was due to

Figure 6. Effect of hibernation on total and phosphorylated levels of Akt in muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii. The upper panel shows representative Western blots. Akt was detected as the dominant band at 60 kDa. Histograms in the lower panel show the normalized band intensities from both conditions (mean Âą s.e.m., n=5). * - Significantly different from the corresponding value for euthermia (P<0.05) [From 129].

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dephosphorylation of the enzyme. In accordance with these data, studies of 13-lined ground squirrels, Spermophilus tridecemlineatus, also demonstrated that Akt phosphorylation and kinase activity were suppressed in liver during hibernation [148]. In summary, Akt enzymatic activity and relative protein expression patterns in ground squirrels agree with data collected from aestivators and hibernating bats described above. Thus, evidence suggests that a pivotal function of Akt regulatory mechanisms during hypometabolism is the inhibition of biosynthetic pathways.

Additional analysis of S. richardsonii muscle Akt showed that the properties of this protein kinase changed significantly between euthermic and hibernating states [129]. In assays at 22°C, S0.5 ATP values (the substrate concentration producing half-maximal enzyme activity) increased by 3.3-fold as compared to the corresponding values for the enzyme from euthermic muscle. The data indicate that Akt undergoes significant changes in its protein-substrate relationships during torpor. This was particularly pronounced for ATP affinity and could have a major effect on suppressing Akt function in vivo during torpor because reduced affinity for ATP during torpor occurs at the same time that ATP availability is also reduced (the total adenylate pool falls by about 30% during torpor). Thus, it may be concluded that there is a reduced enzymatic potential for ATP-dependent phosphorylation of Akt targets during hibernation.

In addition to changes in Akt kinetic properties, the structural stability of the enzyme was analyzed as a possible regulatory mechanism of Akt activity in euthermic versus hibernating states. To evaluate possible differences in structural stability, Akt was subjected to urea denaturation. The I50 values, the concentration of inhibitor that reduces activity by 50%, were 3.51 Âą 0.26 M for control and 2.99 Âą 0.46 M for hibernating conditions, displaying no significant difference. Therefore, urea denaturation experiments concluded that the structural stability of Akt did not change between the two states. Ion exchange chromatography was also used to evaluate the Akt isozyme pattern in muscle. Activity in euthermic control muscle eluted in three peaks indicating the presence of three isozymes: Akt1, Akt2, and Akt3. However, in hibernator muscle, only Akt1 and Akt2 peaks were detected and both of these showed much reduced activity consistent with the general reduction in Akt activity and phosphorylation state described above. Thus, there is evidence that the three Akt isozymes are differentially regulated during hibernation, with Akt3 in particular being strongly suppressed. This may provide a mechanism by which selective regulation of different branches of Akt downstream targets could be achieved when animals enter torpor.

Other studies analyzed Akt responses during the transitory periods of the torpor-arousal cycle (entrance and arousal) rather than deep torpor. During entrance and arousal, which are characterized by high metabolic rates, the

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amount of active Akt increased in liver 13-lined ground squirrels as assessed using immunoblotting [149]. This activation of Akt correlated with the phosphorylation states of two Akt substrates; GSK3β (Ser9) and PRAS40 (Thr246). Phospho-GSK3β levels increased 2.5-fold during entrance into torpor and 2.6-fold during arousal, whereas phospho-PRAS40 levels increased by 4.5-fold in entrance and 4.6-fold in arousal (as compared to control animals). In order to further probe the activation of the mTORC1 pathway, the activation levels of p70-S6K as well as 4EBP1 were also measured. The data identified an upregulation of p70-S6K(T389), a direct target of the mTORC1 complex, during early torpor (Tb=5-8°C for a day) and early arousal (Tb=9-12°C) whereas 4EBP1 showed significant increases in relative protein content during entrance and arousal. Similar to the studies with greater tube-nosed bats, the above data lends support to the hypothesis that intermittent arousal periods back to euthermia during the hibernating season may provide a mechanism to renew cellular stores depleted during deep torpor. Activation of both biosynthetic pathways (through GSK3β) as well as protein synthesis (through mTORC1) during entrance and arousal suggests these processes are important in the liver during hibernation.

Studies of pathways central to the stress response in aestivation and hibernation have eloquently demonstrated mechanisms of Akt regulation.

Typically, Akt is involved in anabolic processes and, thus, it would be expected that Akt is inactivated in order to contribute to energy conservation in the hypometabolic state. However, activated Akt has the capacity to selectively alter multiple downstream targets, such as GSK-3β, mTOR, BAD and FOXO, depending on the need of the organism and differential action by Akt may alter the relative functionality of different target pathways in a hypometabolic state.

For example, the data for aestivating snails indicate that simultaneous inhibition of BAD and FOXO via phosphorylation can occur while leaving GSK-3β inhibition intact, and not stimulating mTOR activity. This could help to ensure that the aestivating land snail can maintain a depressed metabolic state while also inhibiting the detrimental signals that might initiate programmed cell death. In hibernators, Akt is inactivated during deep torpor characterized by low metabolic rates, while transitory phases characterized by higher metabolic rates, such as entrance and interbout arousal, showed activated Akt. Therefore, given the large number of downstream effects observed through Akt signalling; a circumstance-dependent, tissue-specific, and sometimes even organismspecific response is observed.

6.2. Akt and ischemia-reperfusion In addition to its occurrence as part of several well-studied clinical disorders (e.g. heart attack, stroke), I-R (ischemia-reperfusion) is a major

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component of hibernation torpor-arousal cycles. A key challenge for survival of mammalian hibernators is a strong reduction of heart rate and blood flow during torpor, matched with the rapid resurgence of oxygen supplies during arousal. Therefore, hibernating mammals, such as the thirteen-lined and Richardson’s ground squirrels, demonstrate characteristic features of ischemic-reperfusion cycles [150].

Previous studies have shown that the PI3K-Akt pathway plays a critical role in reducing damage during ischemia-reperfusion in multiple tissues. For example, the introduction of a constitutively active mutant form of Akt (myrAkt) protected rat liver tissue from I-R injury through its antiapoptotic effect in phosphorylating BAD [151]. Neuroprotective roles for PI3K-Akt signalling have also been shown in brain, again via antiapoptotic action [152].

PI3K-Akt signalling also appears to exert a similar protective effect in rat heart during ischemic insult [153]. These findings all show the pro-survival role of Akt in protecting organs from I-R induced apoptosis and organ dysfunction.

One major pressure induced by reperfusion is oxidative stress. This occurs due to a surge in reactive oxygen species (ROS) production as a result of a rapid increase in oxygen consumption and mitochondrial activity as hibernating mammals awake from deep torpor. Specifically, the extremely high rates of oxygen uptake and consumption are needed to support nonshivering thermogenesis to rewarm the body. Recent studies have shown that the HSP27 is a co-activator of Akt [126]. HSP27 acts as a scaffold protein for Akt, holding other cofactors to form an activation complex. H2O2 treatment enhanced the interaction between HSP27 and Akt as well as S473 phosphorylation [154]. It also has been reported that Akt was activated in H2O2-treated vascular smooth muscle cells under various conditions [155].

These findings showed that ROS can act upstream of PI3K-Akt activation, indicating a possible protective effect from Akt action in response to oxidative damage.

Transcription factors under the control of Akt regulation may also play a role in the detoxification of ROS. For example, the redox-sensitive transcription factors, NFκB and Nrf2 (i.e. NFE2L2, or nuclear factor (erythroid-derived 2)-like 2), defend cells from oxidative stress by the upregulation of antioxidant enzymes. Studies with the hibernating ground squirrels have demonstrated upregulation of multiple antioxidant enzymes during hibernation including peroxidredoxins, manganese superoxide dismutase and hemeoxygenase under NFκB and Nrf2 control [150, 156-157].

Ground squirrels also show upregulation of HIF-1α which is known to respond to changes in available oxygen [158]. Indeed, HIF-1α protein levels were elevated by 60-70% in the two organs responsive for thermogenesis (brown adipose and skeletal muscle) [158].

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Akt may also impose other regulatory mechanisms on the ischemicreperfusion response through cross-talk with MAPK pathways and, thus, Akt signalling may indirectly mediate this response. For example, MAPKKK family kinases, such as MEKK1 (or MAPKKK1), phosphorylate and activate IKK complexes which, in turn, phosphorylate the IkB protein, the inhibitor of NFÎşB [159]. MAPKs (ERK, JNK and p38) are also involved in the transcriptional activation of Nrf2-mediated gene expression [160], and HIF-1Îą is regulated by ERK2 signalling [158]. Indeed, a sequential activation of a three tiered MAPK cascade involving MAPKKKs (c-Raf, MEKK), the MAPK kinase (MEK1/2), and finally the MAPK (ERK1/2) was documented in response to dehydration in organs of an aestivating frog, Xenopus laevis [161]. In addition, immunoblotting with phospho-specific ERK antibody as well as kinase activity profiles demonstrated that the homologue of p42(ERK2) was increased in snail (L. littorea) foot muscle under anoxia [162], whereas ERK1/2 and the ERK-activated kinase, MAPKAPK-1, rose significantly in muscle and brain during hibernation in Richardson's ground squirrels [163].

Furthermore, the MAPKs, p38 and JNK, showed distinct responses to ischemic insults as compared to ERK2 regulation; (1) phospho-p38 MAPK rose in L. littorea hepatopancreas during anoxia while JNK exhibited no change [144], (2) phospho-p38 activity increased in muscle and heart while JNK activity rose in muscle, heart, liver and kidney in the Richardson's ground squirrels during hibernation [163], and (3) phospho-p38 was activated in skeletal muscle during hibernation of bats, M. lucifugus [164].

7. Conclusion Due to its multiple downstream effects, PI3K-Akt signalling is considered to be one of the most important regulatory signalling pathways of the stress response. As described in previous sections, Akt is generally associated with cell growth through its regulation of metabolism, protein synthesis, cell proliferation, and anti-apoptosis. Akt controls cell proliferation via the activation of cell cycle promoting factors (such as cyclins) and the suppression of the inhibitory factors (e.g. CDKIs). The pathway contributes to managing protein synthesis either following the mTOR-dependent or the eIF2Bdependent mechanisms. Activated Akt can both directly (e.g. phosphorylation of BAD) or indirectly (e.g. inhibitory effect on FOXO transcription factor and p38 signalling) impose negative effects on apoptosis. Its antiapoptotic and prosurvival effects protect cells from death resulting from metabolic perturbations and/or injuries associated with external stresses. In addition, the positive roles in glucose uptake (via promotion of GLUT4 membrane translocation and GLUT1 expression) and glycogenesis (via the inhibition on

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GSK3β and other related factors) make PI3K-Akt signalling pivotal to fuel preservation and energy metabolism.

While the full involvement of PI3K-Akt signalling in response to environmental factors still remains to be fully discovered, a firm connection between the stress response and PI3K-Akt has been proposed by studies in aestivating and hibernating species described in this chapter. In summary, it appears that PI3K-Akt signalling is important in all forms of aerobic dormancies, which sheds some light on its regulatory roles under direct exposure of unfavourable conditions. Therefore, based on past achievements, further investigation can be carried out for a more complete understanding of the stress-responsive mechanism of PI3K-Akt signalling. These will bring us new insights into the biochemical mechanisms behind the physiological adaptations to environmental pressure.

Acknowledgements We thank J.M. Storey for critical commentary and editorial review of the manuscript. In addition, we would like to acknowledge the many students, past and present, as well as collaborators who contributed their data and insights to this review. Research in the Storey lab is supported by a discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to KBS and the Canada Research Chairs program. SNT held a NSERC PGSD scholarship.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates, 2011: 183-202 ISBN: 978-81-308-0471-2 Editors: Anna Nowakowska and Michał Caputa

8. Genetic and epigenetic regulation in hypometabolism Jan Pałyga Department of Biochemistry and Genetics, Institute of Biology, Jan Kochanowski University, ul. Świętokrzyska 15, 25-406 Kielce, Poland

Abstract. Many organisms that periodically encounter extreme environmental conditions evolved adaptations for regulated depression of metabolic rate to survive until the next favorable season. The coordinated metabolic depression is associated with a global inhibition of energy-consuming processes including transcription and translation.

However, both transcription and translation of specific proteins essential for cell protection and survival is maintained albeit sometimes at reduced rate. A limited number of genes, for example those involved in fat oxidation, is up-regulated. Gene transcripts, together with accessory factors, are stored as stress granules in the cytoplasm and become quickly available if needed. Translational silencing of mRNA is partly regulated by specific microRNAs differentially expressed during hibernation as well as hypoxia, which is a hallmark of many hypometabolic states. Global transcriptional silencing could be regulated by epigenetic mechanisms, especially repressive histone posttranslational modifications and chromatin remodeling. Specific upregulation of certain genes during hypometabolism might be associated with a local recruitment of activating epigenetic enzymes and effectors.

Correspondence/Reprint request: Dr. Jan Pałyga, Department of Biochemistry and Genetics, Institute of Biology, Jan Kochanowski University, ul. Świętokrzyska 15, 25-406 Kielce, Poland E-mail: jan.palyga@pu.kielce.pl

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List of symbols and abbreviations AMPK: HDAC: HIF-1: HSP: miRNA (also miR): PGC-1α: SIRT1-7: TCA:

adenosine-5′-monophosphate-activated protein kinase; histone deacetylase; hypoxia-inducible factor 1; heat shock protein; microRNA; peroxisome proliferator-activated receptor γ coactivator-1α; SIR two 1-7, mammalian homologs of yeast SIR2 (silent information regulator 2); tricarboxylic acid;

Introduction Most organisms can thrive not only under permissive but also under unfavorable and harsh environmental conditions of variable duration and degree of severity which usually slow down or even halt normal metabolic processes. These conditions, including lack of food or oxygen, extremes of cold or heat, desiccation or high salinity and many others are often circadian or cirannual in nature and can be encountered several times during a life span of the animal. To survive, the organisms have to cope with the effects imposed by a lack of nutrients, essential elements or compounds and non-physiological range of physical factors which lead to disturbances in the efficiency of pathways responsible for energy and intermediate metabolite production as well as cellular and whole-organism processes guarding integrity of cells, tissues and organs. The most important adaptation commonly employed under most if not all stark environmental conditions is a coordinated suppression of metabolic functions and entering a hypometabolic state in which metabolic rate may be reduced by as much as 70-99% compared with the resting rate [1].

Several common themes underlying hypometabolic states across the animal kingdom have been revealed in natural surviving strategies including hibernation [2-4] and freeze tolerance [5, 6] in cold and freezing seasons, estivation [7] in dry and hot seasons, and hypoxia tolerance [8-10] under fluctuating supply of ambient oxygen. In addition to general suppression of energy production and use, the animals developed strategies to preserve cell structures and function by re-directing metabolites and metabolic energy to processes responsible for supplying factors that prevent or minimize damage to macromolecules by low and high temperatures as well as a surge

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in reactive oxygen species during interbout arousal or resuming a normal metabolism following aerobic recovery from hypoxic/anoxic period in hypoxia-tolerant animals.

Various aspects of common biochemical and cellular changes in hypometabolic states in different species have been reviewed recently [1, 4, 7, 11-17]. The aim of this review is to summarize a contribution of genetic and epigenetic mechanisms to coordinated regulation of metabolism in animals which are evolutionarily well adapted to cope with environmental stresses. In response to inherent seasonal clues and/or external stresses, cellular adaptations involve multiple steps in gene expression including transcriptional, post-transcriptional, translational and post-translational regulation that ultimately lead to either reduced or enhanced production of active proteins, depending on the needs of the cell. These changes in conjunction with allosteric regulation of the enzymes may profoundly affect the rate of metabolism.

Global genetic regulation of transcription and translation While hibernating, estivating or under other forms of dormancy, most animals do not take up food and water and rely mainly on fat stores deposited in the body for energy production which also is severely depressed. Global depression of metabolic rate in species entering hypometabolic states usually involves a suppression of transcription and translation [1, 7, 8]. This process is fully reversible and returns to normal level both during arousal periods in small hibernating mammals and when external environmental conditions are less severe and animals become active. The overall rate of transcription was strongly reduced in tissues of torpid ground squirrels [18] in which a radioactive leucine incorporation into brain proteins was only 0.04% of the mean value of active animals [19]. Under anoxia both protein synthesis and protein degradation were reduced by more than 90% to diminish energy expenditure [20]. Translation is also severely reduced to less than about 0.5% of euthermic values during torpor in ground squirrels [21] and is fully restored during the interbout arousal [22]. Long-term survival in hypometabolic state requires a strong and coordinated suppression of ATP-consuming processes, including â&#x2C6;&#x2019; in addition to transcription and translation â&#x2C6;&#x2019; diminished active transport across membranes and suppressed cell proliferation. Regulated inhibition of gene transcription [1] and ribosomal translation [21, 23] as well as sequestration of mRNA transcripts into storage bodies [24] help to prevent unnecessary protein synthesis and resume translation quickly when organisms exit hypometabolic states.

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In general, it has been proposed [25] that differential gene expression during hibernation is at a heart of seasonal phenotypic differences in physiology of hibernating mammals. Therefore, a capacity to survive during severe seasons may rely to a great extent on regulated and coordinated use of common genes rather than specially evolved ones. Nevertheless, amino acid sequence differences which may enhance performing specific function under long-term stressful conditions were also noted [26]. The amino acid sequence of phosphoglycerate kinase 1, the essential glycolytic enzyme involved in ATP synthesis under anaerobic state, is highly conserved across vertebrate phylogeny. However, the phosphoglycerate kinase 1 of freeze-tolerant wood frog (Rana sylvatica) harbors unique amino acid substitutions in the positions which were fully conserved in other species. It is possible that these substitutions may furnish freeze-tolerant frog with a flexibility needed to maintain an active conformation of the enzyme over the wide range of temperatures which the animal encounters in its natural habitat [26].

Both genome-wide and proteomic comparisons using microarrays [27] and liquid chromatography followed by tandem mass spectrophotometry [28] for relative levels of specific transcripts and proteins, respectively, in hibernating arctic ground squirrels (Urocitellus parryi) have revealed significant seasonal changes in metabolic gene expression and protein content in multiple tissues (brown adipose tissue, liver, heart, hypothalamus and skeletal muscle). In general, under-expression of glycolytic and over-expression of fatty acid catabolic genes [27] was consistent with a paradigm that energy catabolism shifts from carbohydrate substrates to fatty acids during hibernation [2]. The seasonal differences in global gene expression were more significant than variations in gene transcription during the torpor-arousal cycle [27]. Proteomic data for arctic ground squirrels [28] were consistent with the gene expression studies both in this species [27] and black bear (Ursus americanus) [29], supporting a significant down-regulation of enzymes involved in glycolysis, fatty acid and lipid biosynthesis, cellular respiration and urea cycle, and upregulation of genes for gluconeogenesis, fatty acid β-oxidation and ketone body metabolism during hibernating season. The proteomics results [28] also confirmed the observations of the gene expression study [27] that expression from a subset of metabolic genes decreased during the transition from late torpor to early arousal and that circadian rhythm and cell cycle were suspended during torpor and resumed during arousal.

Seasonal proteomic changes were also detected in liver of hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) using fluorescent dye-stained two-dimensional gels followed by liquid chromatography and tandem mass spectrometry [22]. This research confirmed a shift from oxidation of dietary carbohydrates in summer animals to oxidation of stored

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fat during hibernation for energy production as evidenced by a decrease in key catabolic enzymes of carbohydrates and increase in enzymes involved in fatty acid catabolism throughout the winter. Many metabolic enzymes that decreased in winter were directly or indirectly linked with tricarboxylic acid (TCA) cycle. For example, a 4-7.2-fold decrease in three spots corresponding to ATP citrate lyase in torpid animals, a result confirmed by Western blotting, can be explained by a general decrease in fatty acid biosynthesis during hibernation [22]. In summer, the ATP citrate lyase provides acetylCoA for fatty acid biosynthesis. Moreover, increased levels of enzymes involved in pentose phosphate pathway in summer may reflect a need for supplying NADPH for reductive stages in fatty acid biosynthesis. The levels of enzymes involved in amino acid catabolism, for example glutamateoxaloacetate transaminase, glutamate-pyruvate transaminase and urea cycle enzymes, were reduced to conserve amino acids during winter hibernation.

Metabolomic profiling of plasma samples collected during natural stages of yearly cycle of the circannual hibernator, thirteen-lined ground squirrel, revealed a number of metabolites that alter with season and hibernation stages, including specific free fatty acids, antioxidants and modified amino acids [30]. As the major pattern in metabolite levels consisted of either depletion or accrual during torpor followed by a return to homeostatic levels by interbout arousal, it has been concluded [30] that these results support a hypothesis that energetically-costly periodic arousals are necessary to restore metabolic homeostasis via mechanisms that only operate at euthermic temperatures. Accumulation of N-acetylated and Îł-glutamylated essential amino acids seems to indicate the presence of salvage mechanism that spares and recycles essential amino acids for use in new protein synthesis during winter fasting [30]. Protein synthesis in hibernators is shut off during torpor [2] and restored to summer levels during each interbout arousal [22]. Periodic protein biosynthetic capability throughout the winter may allow the cells of aroused hibernators to replenish the proteins that were damaged or depleted during torpor [31]. The proteins that increased in winter were mainly confined to those involved in protein synthesis, folding, transport and stability, as well as RNA-interacting proteins and other proteins performing specialized functions. For example, a relative cardiac myosin heavy chain-Îą protein expression increased significantly in the left atrium during hibernation of grizzly bears (Ursus arctos horribilis) to protect the heart from excessive work against optimally filled ventricle [32]. Though active protein biosynthesis may lead to increased energy consumption, this process can be controlled by posttranslational mechanisms, such as phosphorylation of initiation factors involved in translation [33], to inactivate reversibly key components during torpor [19]. Reversal of inactivating modification during

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arousal could allow rapid resumption of protein synthesis during each interbout arousal [22] when shivering and non-shivering thermogenesis is active, and consumption of oxygen and ATP synthesis is high.

The brains of hibernating ground squirrels are markedly resistant to oxidative damage as they undergo transitions in activity during the torpor and arousal cycles in winter [34]. The seasonal alteration in protein composition was measured by the changes in protein spot densities observed between summer and winter brainstem [35], a region containing neurons of the autonomic nervous system with critical functions for survival during hibernation. Compared to proteomic alterations in other tissues [22, 28, 36], the changes in brainstem proteins were less pronounced [35] demonstrating that majority of the protein complement remained unaltered between summer and torpid animals. This feature is consistent with a need to support and maintain the brain cell functions throughout the year. A transition from carbohydrate catabolism to lipid oxidation during hibernation resulted in a decrease of many enzymes of glycolysis and increase in abundance of the enzymes involved in alanine and aspartate metabolism, TCA cycle and oxidative phosphorylation in the brainstem.

Up-regulation of numerous mitochondrial proteins appears to suggest that periodic ATP synthesis during hibernation may involve a selective use of initial reactions of TCA cycle. At the end of euthermic interbout arousal the elevated level of ATP could be used to phosphorylate key TCA cycle enzymes, for example Îą-ketoglutarate dehydrogenase, leading to inhibition their activities and reduction in ATP synthesis. The TCA cycle substrates derived from ketone bodies and amino acid metabolism would accumulate in the mitochondrial matrix during torpor because the TCA enzymes are inactive at that time [35]. At the end of torpor when body temperature is still low and ATP reserves are depleted [37] the phosphorylated enzymes (Îą-ketoglutarate, isocitrate and pyruvate dehydrogenases) are activated by dephosphorylation and ready for generation of reduced coenzyme, NADH, which is then reoxidized by electron transport chain located in the inner mitochondrial membrane. To this end, a relatively high copy number of proteins in oxidative phosphorylation pathway is needed for rapid production of ATP during arousal when demand for energy is high [35].

Transcription factors, coordinated gene expression, and cell survival in hibernators Another approach to understanding gene regulation that supports hypometabolic phenotype is to identify the transcription factors that are

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activated when animals enter torpor or other metabolically depressed state [1]. Typically, a particular transcription factor regulates a group of genes that are performing a specific cell function, and therefore by identifying specific transcription factors it is possible to dissect molecular events underlying phase-specific or organ-specific adaptation over the torpor–arousal cycle. In overall, there are two main aspects involved in the regulation of hypometabolic states [1]. The first is a set of mechanisms that regulate and coordinate metabolic functions associated with transitions between various hypometabolic phases, such as entering torpor or entering arousal from torpor. This type of control can be accomplished by enzyme regulation through a reverse post-translational protein modification, activation of stored mRNA templates or transcription of a limited number of genes. A second important facet of torpor is linked to cell preservation mechanisms that maintain viability over days, weeks, or months in a torpid or dormant state.

For example, it has been recently shown [38] that active regulation of the skeletal muscle transcription factors, myocyte enhancer factors 2A and 2C, occurs over a torpor-arousal cycle in thirteen-lined ground squirrels. The level of these proteins rose nearly threefold during the torpor phase, and the amount of phosphorylated active myocyte enhancer factors, which are targeted to the nucleus, increased by half. The actions of these transcription factors through regulation of selected target genes may preserve muscle mass during long-term cold torpor and support shivering thermogenesis in skeletal muscle during arousal [38].

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that is expressed across metazoan phylogeny and functions as a master regulator of oxygen homeostasis [39]. Up-regulation of the α-subunit of this factor (HIF-1α) also occurs in thermogenic organs (brown adipose and skeletal muscle) of ground squirrels during torpor [40]. Both HIF-1α protein level and its DNA binding capability in the nuclear extracts from brown adipose were higher during torpor. HIF regulates a large number of genes by binding to a cis-acting hypoxia response element within target gene promoters. In response to hypoxia, the genes show either increased or decreased HIF-1dependent expression. The mechanism by which HIF-1 binding leads to transcriptional activation or repression presumably involves the recruitment of coactivators or corepressors [39], respectively. It has been hypothesized [1] that HIF-1 may exert an inhibitory action during hibernation by suppressing the expression of genes involved in the cell cycle and other ATPexpensive functions, and that it could mediate selective remodeling of some subunits of the electron transport chain to allow for more efficient use of oxygen. Optimization of cytochrome oxidase function would be important for the performance of hibernator thermogenic tissues. In fact, an up-regulation

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of subunits of cytochrome c oxidase and NADH-ubiquinone oxidoreductase was detected in the hibernators [41, 42].

Enhanced antioxidant defense is a universal feature of hypometabolism [43] and was detected in hibernating ground squirrel [44] and turtle hatchling [45, 46], during dehydration stress in frog [47], and estivation in snails [48, 49]. Efficient antioxidant defense is essential to provide the protection of macromolecules against a damage by reactive oxygen species in the hypometabolic state when replacement of damaged macromolecules by new synthesis is very restricted, and to defend against a rapid rise in reactive oxygen species production when oxygen uptake and consumption increases rapidly during arousal. At these circumstances, there is a variable up-regulation of antioxidant enzymes including superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, peroxiredoxins as well as a redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 [1]. A strong increase in the levels of the nuclear factor erythroid 2-related factor 2 and its heterodimeric partner, MafG (v-maf musculoaponeurotic fibrosarcoma oncogene homolog G), and increased nuclear translocation of the factor erythroid 2-related factor 2 and MafG heterodimer were observed [50] in several tissues of torpid thirteenlined ground squirrels. This effect was accompanied by the up-regulation of the erythroid 2-related factor 2 gene targets: heme oxygenase transcript and protein levels rose in liver, kidney, heart and brain [50] while Cu/Zn superoxide dismutase, aflatoxin aldehyde reductase and peroxiredoxin isozymes were elevated in the heart [51] of torpid animals. Peroxiredoxins, stress responsive proteins engaged in reducing and detoxifying of a range of hydroperoxides using thioredoxin as an electron donor, have been suggested [1] to play a key role in intracellular antioxidant defense in mammalian hibernation.

Animals experiencing environmental stress, including those that relapse periodically into hypometabolism, developed efficient defending mechanisms against damage to cellular proteins by up-regulating chaperone proteins that assist in both folding of nascent proteins and refolding of unfolded or malfolded proteins as a result of severe and long-lasting stresses. It has been shown [52] that anoxic submergence in fresh water turtle Trachemys scripta elegans activated heat shock transcription factor 1. In response to stress, heat shock factor 1 undergoes hyperphosphorylation to promote trimerization and translocation into nuclei to induce gene expression of heat shock proteins (HSP) by binding to cognate elements in their promoters. Although enhanced nuclear localization of active form of HSP1 was observed in all tested turtle tissues (heart, kidney, liver and muscle), the pattern of HSP induction was organ-specific. In particular, HSP60, a mitochondrial chaperone, was

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elevated only in liver, Hsp40 and Hsc70 (heat shock protein 70 cognate) displayed a correlated response to anoxia in all tissues examined while none of the heat shock proteins examined was elevated in heart [52]. The upregulation of heat shock proteins during anoxia indicates important role for these chaperone proteins in maintaining proper conformation of proteins under depressed rate of metabolism.

MicroRNAs and hypometabolism

post-transcriptional

regulation

in

A large portion of genome is transcribed into non-coding RNAs [53].

Many small non-coding RNAs, known as microRNAs (miRNAs), are involved in post-transcriptional regulation [54] mainly through lowering the level of mRNAs [55] and/or suppression of their translation [54]. Moreover, some miRNAs and other non-coding RNAs may be involved in epigenetic regulation [56] by controlling the expression of components of the epigenetic machinery [57], guiding modifying enzymes to specific loci in the genome [58], and RNA-RNA and RNA-protein interactions [53, 59].

Genes encoding miRNAs are initially transcribed by RNA polymerase II into long primary miRNA transcripts which are first processed in nuclei by type III RNA endonuclease Drosha into pre-miRNAs that are much shorter and form stem-loop structures [60]. After transportation into the cytoplasm by exportin-5, pre-miRNAs are further processed by another type III RNA endonuclease, Dicer, which generates ~22-nucleotide miRNA duplexes.

Mature single-stranded miRNAs form ribonucleoprotein complexes, RNAinduced silencing complexes, containing a core protein component belonging to Argonaute protein family in a process which requires ATP for assembly [61]. This complex regulates post-transcriptional fates of mRNAs by base pairing between the nucleotides 2-8 of the miRNA and 3â&#x20AC;˛ untranslated region of its target genes [54]. The extent of miRNA-mediated gene regulation is potentially high due to ability of individual miRNA to target transcripts of many genes and capability of the same mRNA to be targeted by multiple miRNAs which ultimately may generate a huge number of regulatory interactions allowing for reversible fine tuning of the actual translation [11].

Since miRNAs control numerous aspects of cell functions, they are also implicated in suppressing metabolism, protein synthesis, cellular proliferation and apoptosis under hypometabolic conditions [11]. Differential expression of miRNA species as well as elevated amount of Dicer, enzyme involved in miRNA processing, were detected [62] in several tissues (kidney, skeletal muscle and heart) of thirteen-lined ground squirrels during torpor. Using

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reverse transcriptase polymerase chain reaction amplifications, the abundance of two miRNAs, miRNA-16 and miRNA-21, were evaluated in liver and skeletal muscle during freezing of wood frog (Rana sylvatica). While miRNA-21 levels increased significantly in liver and muscle, the miRNA-16 transcripts rose significantly only in liver and fell by 50% in muscle of the frozen frogs. In model systems of hypoxia, the induction of HIF-1α was accompanied by induction of several miRNAs [63, 64] of which increase in miRNA-210 level was consistent and robust. The HIF-1α-mediated induction of miRNA-210 was implicated in regulating genes required for angiogenesis, cell proliferation, cell differentiation including erythropoiesis, DNA damage repair, apoptosis, and mitochondrial metabolism [63]. Upon hypoxia, up-regulated miR-210 targets iron-sulfur cluster assembly proteins that are critical for electron transport in mitochondria and thereby shuts down aerobic mitochondrial energy generation and switches the hypoxic cell to anaerobic metabolism [65]. The miRNA-210 is an example of a large group of stressresponsive miRNAs involved in the control of gene expression under unfavorable conditions [66] that are invariably encountered in most stressed cells.

More than 200 miRNA species have been identified [67] in the course of genomic analysis of miRNAs in Arctic ground squirrel (Urocitellus parryi) using Illumina deep-sequencing, Agilent miRNA microarray and real-time polymerase chain reaction. Some miRNAs, for example miR-320 and miR-378, were significantly under-expressed during hibernation compared with summer animals, whereas others, like miR-451 and miR-48, were overexpressed in late torpor and returned in early arousal to the levels similar to those in non-hibernating animals. As the miRNA targets may be affected both at the mRNA and protein levels, the Pearson correlations were computed [67] between the levels of miRNA expression [67] and putative target gene expression at the mRNA level from real-time PCR results [28] and Illumina BeadArray assay results [27], as well as between miRNAs [67] and differently expressed proteins from proteomic study [28], employing the fact that these experiments were conducted on the same set of animals. These comparisons revealed [67] a negative correlation between miRNAs and the gene expression at both protein and mRNA levels for some putative target genes. However, specific functions of differentially expressed miRNAs during hibernation of Arctic ground squirrels remain largely unknown.

Total mRNA transcript levels do not change significantly over torporarousal cycle during hibernation [68, 69] and, thus, these transcripts appear to be stabilized in an inhibited form during torpor to became immediately available for translation when the animal arouses [4]. The majority of transcripts are stored during torpor in a translationally silent monosome

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fraction [19, 70] so that only transcripts that are actually translated during this phase, for example transcripts for fatty acid binding protein, remain associated with polysomes [42]. In addition, mRNAs harboring internal ribosome entry site elements isolated from livers of golden-mantled ground squirrels [23] preferentially associate with ribosomes as torpor bout progresses which allows for immediate production of specific proteins when the squirrels arouse from torpor and translation resumes.

The stress response which is often associated with inhibition of translation initiation [71, 72] leads to the formation of cytoplasmic RNA-protein complexes referred to as stress granules [24, 73]. Stress granules contain nontranslated mRNAs, translation initiation factors and accessory proteins implicated in the control of mRNA function. During recovery from stress, the stress granules disassemble to resume translation of individual mRNAs [73].

The cytoplasmic RNA granules, such as processing bodies (P-bodies) and stress granules, are important for posttranscriptional regulation of gene expression. The P-bodies which contain components of mRNA decay machinery are often juxtaposed close to stress granules whose main function is a storage of untranslated mRNAs. Argonaute proteins, core components of miRNA-induced silencing complexes, have been shown to be localized both in P-bodies and stress granules [74]. It is possible that under stress conditions miRNA-induced silencing complex-bound mRNA is first targeted to stress granules for translational repression and temporal storage, and then â&#x2C6;&#x2019; following a sorting step â&#x2C6;&#x2019; is delivered to the P-bodies for decay [75].

Epigenetic control of gene expression The meaning of epigenetics, a term coined by Conrad Waddington [76, 77] to describe a process of cellular decision making during development, has changed over the time and now is often used to describe a heritable transfer, usually mitotic, of phenotypic characters without modification of the genetic code of underlying gene sequences [77]. Epigenetic mechanisms permit functional specialization among the cells containing essentially the same genetic information. Specific modifications to histone proteins and DNA produced by enzymes (the 'writers') are recognized by other proteins and protein complexes (the 'readers'). These modifications can be removed by specific enzymes (the 'erasers') if needed. The epigenetic changes in chromatin control how the genome is accessed in different cell-types under varying physiological and environmental conditions. Adaptation to environmental changes as well as cell differentiation and specialization require a coordination of transcriptional output of the genome. Many transcriptional responses are self-sustaining and self-propagating across the cell progeny in the absence of

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triggering stimuli and they constitute a molecular memory of past events that is usually stored in the form of DNA methylation, histone and non-histone protein modifications or non-coding RNAs. These transcriptional states may constitute (i) trans epigenetic states in which epigenetic signals are maintained by feedback loops and networks of transcriptional factors and some non-coding RNAs, and (ii) cis epigenetic states in which epigenetic signals are physically associated and inherited with the chromosome on which they act, for example DNA methylation or histone modifications [78]. Histones may carry epigenetic information in their primary sequence (histone variants) [79], in posttranslational modifications (acetylation, methylation, phosphorylation, ubiqitylation, sumoylation), especially in their N- and C-terminal tails [80], or in their position relative to DNA sequence following sliding [81]. In addition, cis epigenetic information may also be conveyed by chromatin non-histone proteins, higher order chromatin structures and nuclear localization of the chromatin regions [78, 82].

There are three categories of signals that culminate in the establishment of a stable heritable epigenetic state [83]. External signals coming from outside of the cell, such as differentiation signals, environmental exposure and nutrition, can be considered as an “epigenator signal”. Once such a signal is received, it may be transmitted through protein-protein interactions to become converted into an intracellular “epigenator” signaling pathway. An “initiator” translates the external signal to establish chromatin context at a precise genomic location. It determines when and where epigenetic modification will occur. The “initiator” can be a DNA-binding protein (e.g. transcription factor) or a non-coding RNA. Unlike the “epigenator”, the “initiator” may not dissipate after its action, but rather may persist with a “maintainer” whose main task is to sustain the resulting epigenetic chromatin state. The maintainer may recruit many different chromatin components such as histone and DNA modifiers (enzymatic activities that convey modifications; [80]), non-canonical histone variants replacing the canonical variants synthesized during S-phase of the cell cycle [79] or nucleosome repositioning molecular machines [84, 85]. The chromatin modifiers may operate at any chromosomal location to which they are recruited by the “initiator” [83].

Repressive epigenetic modifications include, for example, DNA methylation [86] or histone H3 K9 and K27 methylation [80] while activating modifications are represented by H3 and H4 lysine acetylation or H3 K4 methylation [80]. The histone modifications are recognized by specific structural folds in effector proteins [87] which facilitate recruitment of multisubunit complexes to target chromatin sites to suppress or enhance transcription. Histone modifications are reversible so that acetyl moieties

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introduced by histone acetylases can be detached by deacetylases [88], and methyl marks left by histone metylases are erased by specific demethylases [89]. Even DNA methylation marks can be specifically removed by active DNA demethylation [90].

Epigenetic alterations in hypometabolism Despite profound changes in transcriptional activity when the animal enters seasonally- and environmentally-induced depression in metabolic state, the contribution of epigenetic mechanism to reversible alterations in gene expression during hypometabolism is relatively poorely explored. A high transcriptional activity is generally correlated with high acetylation levels of histones, especially H3 and H4, in nucleosomes associated with transcribed loci whereas repression of transcription is accompanied by a reverse process of histone deacetylation [91]. Reduced transcriptional state during thirteen-lined ground squirrel hibernation was found to be linked with (i) a decrease of histone H3 phoshorylation at Ser10 and H3 Lys23 acetylation levels in muscle by more than one third and (ii) significant increases in the expression of histone deacetylases HDAC1 and HDAC4, and total HDAC activity [92]. The phosphorylation of histone H3 at Ser10 residue is usually associated with mitotic chromosomes, however, phosphorylation at this site may occur at some transcriptionally active chromatin regions as well [93]. It seems that diminished phoshorylation at H3 Ser10 may reflect a tendency toward the cell cycle arrest under unfavorable conditions [94]. Histone deacetylases are unable to recognize directly specific sites which have to be deacetylated and need to be recruited by co-repressors to their sites of action [95]. A level of expression of transcriptional corepressor SIN3 (suppressor interacting 3) homolog A which interacts with HDAC1 [96] was found to increase significantly in muscles of 6-month estivating Australian frog Cyclorana alboguttata [97]. These frogs exhibited also a significant up-regulation of DNA cytosine-5-methyltransferase 1 [97], a maintaining enzyme involved in epigenetic methylation of CpG dinucleotides [98] in promoter sequences to attenuate transcription from the respective genes [86]. DNA methylation is rather a stable epigenetic modification but at some promoters, including estrogen-responsive pS2 gene, a cyclical CpG methylation and active demethylation of 5-methylated CpGs was reported [99]. Thus, it is possible that a long-term metabolic rate depression might have involved a methylation of promoter DNA sequences at specific genes which could be reversed upon returning to active life.

Although DNA methylation and gene expression are responsive to caloric restriction in humans [100], currently there is a lack of direct evidence for a

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link between the extent of DNA methylation and metabolic depression in free-living animals.

Transcript and protein level of class I and class II histone deacetylases, HDAC1-3 and HDAC4-5, respectively, rose significantly in white skeletal muscle upon 20-h submergence of freshwater turtle Trachemys scripta elgans at 4°C [101]. The total activity of HDACs in the muscle increased by 1.5-fold while the levels of histone H3 acetylated at Lys9 and Lys23 decreased by approximately 1.7-2.5-fold. Less prominent increases in the HDAC protein levels were observed in liver of hypoxic turtles but the levels of acetylated histone H3 in this tissue also decreased to 50-75% of control values. In the heart of anoxic turtles only HDAC5 responded to low oxygen level by increasing its transcript and protein abundance. This protein, however, does not play a major role in transcriptional silencing because the levels of acetylated histones in the heart did not change significantly in response to anoxia. Heart remains active throughout hypoxia/anoxia period since its action is crucial for surviving by providing nutrients to and removing wastes from all the organs of hypoxic turtle [101].

A link between histone and chromatin modifying enzymes and cell metabolism A growing body of data indicates that chromatin enzymatic activities involved in epigenetic modifications are finely and specifically regulated by a variety of small molecules derived from the intermediary metabolism. Both histone- and non-histone chromatin protein-modifying enzymes and ATPdependent chromatin remodeling complexes are regulated by evolutionary conserved coenzymes and metabolites such as adenosine-5′- triphosphate, S-adenosylmethionine, acetyl coenzyme A, nicotinamide adenine dinucleotide, flavin adenine dinucleotide, α-ketoglutarate and inositol polyphosphates. These modifying enzymes can therefore link metabolism to transcription [102-105] and indirectly to environmental changes through the relationship between their abundance and factors influencing the rate of metabolism, including availability of food and oxygen.

In contrast to single-cell eukaryotes that largely rely on acetyl-CoA synthetase to produce acetyl-CoA from acetate, metazoans use glucose as their carbon source. Wellen and colleagues [106] have shown that histone acetylation in mammalian cells is dependent on adenosine triphosphatecitrate lyase, an enzyme that converts glucose-derived citrate into acetyl-CoA not only in the cytoplasm but also in the cell nuclei. The adenosine triphosphate-citrate lyase-dependent production of acetyl-CoA and increase in histone acetylation are tightly coordinated with cellular metabolic state.

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Increased availability of acetyl-CoA, a substrate for histone acetyltransferases, will lead to enhanced acetylation at specific lysines in N-terminal histone tails and subsequent up-regulation of gene transcription to provide key metabolic enzymes. Histone deacetylases catalyze removal of acetyl moieties from ε-amino groups of specific lysine residues in histones and other regulatory proteins rendering the affected chromatin region recalcitrant for transcriptional initiation [88]. Suppression of metabolic rate, is associated with enhanced histone and protein deacetylase activities, especially sirtuins (silent information regulator 2 proteins) which utilize NAD+ as a co-substrate [105], and act as metabolite sensors because their activity depends on the ratio of [NAD+]/[NADH] [107].

Increasing evidence suggests [108, 109] that sirtuins play important role in maintaining serum glucose concentration in mammals within a narrow range under a variety of physiological conditions, including starvation, through regulation of hepatic gluconeogenesis. The ability of coactivator PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α) to modulate gluconeogenesis and fatty acid oxidation pathways in the liver appears to require the opposing actions of SIRT1 deacetylase and the GCN5 (general control of amino-acid synthesis 5) acetyltransferase [109, 110]. The observation that SIRT6-deficient mice demonstrate severe hypoglycaemia suggests a potential role for other sirtuins in glucose production and homeostasis [111]. In addition to regulating hepatic gluconeogenesis, sirtuins also modulate serum glucose levels by regulating pancreatic insulin secretion.

Other metabolic effects include SIRT4-dependent ADP-ribosylation of glutamate dehydrogenase and interactions with insulin degrading enzyme [109], and urea cycle regulation [112]. SIRT1 through its regulation of peroxisome proliferator-activated receptor γ and PGC-1α activity also has a significant regulatory role in fat mobilization and fatty acid oxidation [113].

Since PGC-1α is a key regulator of mitochondrial biogenesis [114] and there is a link between SIRT1 and autophagy [115], it seems that sirtuins might regulate the flux of mitochondria within cells by balancing PGC-1α-mediated production with autophagy-dependent clearance [109]. Moreover, SIRT3 regulates mitochondrial fatty-acid oxidation [116] and ketone body production [117] by reversible deacetylation of respective enzymes.

Adenosine-5′-monophosphate-activated protein kinase (AMPK), a central regulator of cellular energy homeostasis in mammalian cells [118], was found to activate transcription through direct association with chromatin and phosphorylation of histone H2B at serine 36 [119]. AMPK recruitment and H2B Ser36 phosphorylation co-localized within genes activated by AMPK-dependent pathways, both in promoters and in transcribed regions.

Ectopic expression of H2B S36A mutant in which Ser36 was substituted by

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alanine reduced transcription and RNA polymerase II association to AMPKdependent genes, and lowered cell survival in response to stress. Thus, AMPK-dependent H2B Ser36 phosphorylation may represent a general stress-response event in transcriptional and chromatin regulatory pathway leading to cellular adaptation to stress [119].

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Conclusions and perspectives This book shows that hypometabolism is a complex phenomenon involving profound shifts at all levels of the life-organization, ranging from molecular (genetic) to whole-organism (behavioural) adaptive responses.

After I had read manuscripts of all chapters of the book I realised that one important topic is missing: there is no chapter dealing with hypometabolism in fish, in general, and in lungfish exposed to long droughts, in particular.

This topic, to the best of my knowledge, is markedly less explored than those presented in the book.

There are still many unanswered, or only partly answered questions concerning hypometabolism. One of them concerns endogenous factors triggering entrance to and arousal from hypometabolism. A couple of the unanswered questions concern the role of the central nervous system in the control of hypometabolism, as well as its participation in hypometabolic effector responses. There is a scarcity of electrophysiological data on activity of specific neural pathways over hibernation/activity cycles. Shifts in 14C-2deoxyglucose uptake throughout the cycles in the ground squirrel brain suggest selective activation of few micro-regions of the brain-stem during the hypometabolic state. The next unanswered question is association of a specific winter reduction in cranial volume and brain weight in shrews and voles (so called Dehnelâ&#x20AC;&#x2122;s effect) with cerebral hypometabolism. The reduction is a consequence of the seasonal loss of some neurons and many synaptic connections. Cerebral hypometabolism in hypoxic mammals and birds is also linked to activity of selective brain cooling mechanism.

Therefore, continuation of extensive research concerning this defence mechanism is necessary. Altogether, some organ-specific adaptive responses should be the target of the future hypometabolism research.

Besides scientific perspectives of the hypometabolism research its practical aspects are also important. Various components of the hypometabolic responses discovered in animals are of great significance to medical practice.

This concerns not only perspectives of treatment of asphyxiated babies (to maintain them under hypometabolic state) and rescuing the victims of accidental deep hypothermia (lessons from mammalian hibernation should be applied to elaborate methods of safe, highly coordinated rewarming of the human body). Artificial hibernation in humans is also an intriguing target. The future research of hypometabolism in aquatic turtles should shed new light on the problem of longevity and negligible senescence, which are the subject of eternal dreams of the mankind.

MichaĹ&#x201A; Caputa