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207 Biol. Rev. (2004), 79, pp. 207–233. f Cambridge Philosophical Society DOI : 10.1017/S1464793103006195 Printed in the United Kingdom Metabolic rate depression in animals: transcriptional and translational controls Kenneth B. Storey* and Janet M. Storey College of Natural Sciences, Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6 (E-mail : [email protected]) (Received 6 June 2002 ; revised 28 March 2003; accepted 14 April 2003) ABSTRACT Metabolic rate depression is an important survival strategy for many animal species and a common element of hibernation, torpor, aestivation, anaerobiosis, diapause, and anhydrobiosis. Studies of the biochemical mechanisms that regulate reversible transitions to and from hypometabolic states are identifying principles of regulatory control that are conserved across phylogenetic lines and that are broadly applied to the control of multiple cell functions. One such mechanism is reversible protein phosphorylation which is now known to contribute to the regulation of fuel metabolism, to ion channel arrest, and to the suppression of protein synthesis during hypometabolism. The present review focuses on two new areas of research in hypometabolism : (1) the role of differential gene expression in supplying protein products that adjust metabolism or protect cell functions for long-term survival, and (2) the mechanisms of protein life extension in hypometabolism involving inhibitory controls of transcription, translation and protein degradation. Control of translation examines reversible phosphorylation regulation of ribosomal initiation and elongation factors, the dissociation of polysomes and storage of mRNA transcripts during hypometabolism, and control over the translation of different mRNA types by differential sequestering of mRNA into polysome versus monosome fractions. The analysis draws primarily from current research on two animal models, hibernating mammals and anoxia-tolerant molluscs, with selected examples from multiple other sources. Key words : hypometabolism, metabolic rate depression, hibernation, anoxia tolerance, aestivation, protein synthesis, reversible phosphorylation, gene expression, polysome profiles. CONTENTS I. Introduction ................................................................................................................................................. II. Coordinate regulation of metabolic rate depression .............................................................................. (1) Reversible phosphorylation regulation of metabolic enzymes ........................................................ (2) Reversible phosphorylation regulation of membrane ion channels and receptors ...................... III. Metabolic arrest and suppression of protein synthesis ........................................................................... (1) Control of translation by reversible protein phosphorylation ......................................................... (2) Ribosome aggregation state ................................................................................................................. (a) Differential distribution of individual mRNA species ................................................................ (b) Anticipatory up-regulation ............................................................................................................. IV. Metabolic arrest and suppression of protein degradation ..................................................................... V. Metabolic arrest and gene expression ...................................................................................................... (1) RNA synthesis ....................................................................................................................................... (2) Gene discovery ...................................................................................................................................... (3) Hypometabolism-induced gene expression ....................................................................................... (4) Hibernation-induced gene expression ................................................................................................ * Address for correspondence. 208 209 210 210 212 213 216 218 220 221 221 221 222 223 223 Kenneth B. Storey and Janet M. Storey 208 (a) a2-Macroglobulin ............................................................................................................................ (b) PDK4 ................................................................................................................................................ (c) Myosin .............................................................................................................................................. (d ) Fatty acid binding proteins ............................................................................................................ (e) Uncoupling proteins ........................................................................................................................ (5) Anoxia-induced gene expression ......................................................................................................... (a) Gene expression during anoxia in turtles ..................................................................................... (b) Gene expression during anoxia in marine snails ........................................................................ (c) cGMP mediation of anoxia-induced gene expression ................................................................ VI. Conclusions .................................................................................................................................................. VII. Acknowledgements ...................................................................................................................................... VIII. References .................................................................................................................................................... I. INTRODUCTION The ability to suppress metabolic rate strongly and sink into a hypometabolic state is a life-saving mechanism for many organisms. When animals are faced with environmental stresses that threaten normal life, limit food availability or impose severe challenges to their physiology, they can retreat into a hypometabolic or dormant state where they remain until conditions are again conducive to active life. Examples of hypometabolism abound as animal responses to high and low temperature, oxygen deprivation, food restriction and water limitation (Storey & Storey, 1990). In mammals and birds, hypometabolism ranges from nightly torpor in many small species to winter hibernation that can last as long as nine months for some mammals living in cold Arctic or alpine habitats (Geiser, 1988 ; Wang & Wolowyk, 1988). Aestivation allows many animals, including lungfish, frogs, toads and snails, to survive through long dry seasons (Pinder, Storey & Ultsch, 1992; Abe, 1995; Land & Bernier, 1995) whereas diapause arrests the developmental cycle of many insects and other animals to permit survival through harsh conditions and/or coordinate transitions to the next life stage among the many individuals in a population (Danks, 1987 ; Hairston & Caceres, 1996; Podrabsky & Hand, 1999; Denlinger, 2001). Many organisms are excellent facultative anaerobes and one of the keys to survival of long-term oxygen deprivation is metabolic rate depression that lowers tissue energy demand to a level that can be supplied by pathways of fermentative ATP production alone (Storey, 1993; Grieshaber et al., 1994; Lutz & Storey, 1997; Hochachka & Lutz, 2001; Jackson, 2001). In most of the cases of hypometabolism cited above, metabolic rate is lowered to between 5 and 40 % of the resting rate of the normal animal and most experience little or no change to the internal environment of their cells and organs so that arousal can be rapid when favourable environmental conditions return (Guppy & Withers, 1999). Hypometabolism can also be taken to extremes by cryptobiotic organisms (including many seeds, spores, cysts, and eggs). In these metabolic rate is typically less than 5% of normal and, in many cases, a virtually ametabolic state exists (Crowe, Hoekstra & Crowe, 1992 ; Guppy & Withers, 1999; Clegg, 2001). Such organisms experience a profound change in their internal environment often involving extreme dehydration and the addition of high levels of protectants to 224 224 224 225 225 226 226 226 228 228 228 228 stabilize cellular macromolecules for periods of dormancy that can extend to many years. Studies of hypometabolism in nature have been, and still are, episodic ; both physiological and biochemical studies have progressed to different stages in different animal models and the primary focus of research is often quite different with each animal model. Much of the early work with all models centred in two areas : (1) quantifying physiological parameters of metabolic suppression including the percentage reduction in metabolic rate and factors such as bradycardia, apnoeic breathing patterns, hypercapnia, and acidosis that are common elements of hypometabolism across phylogeny, and (2) analyzing changes in the patterns of fuel use, end product accumulation, and the biosynthesis of the low molecular weight protectants that are present in various systems. More recently the focus of much research has shifted to the biochemistry of metabolic rate depression and studies with multiple systems (e.g. hibernating mammals, anoxia-tolerant molluscs and turtles, estivating amphibians and snails, diapausing insects, anhydrobiotic brine shrimp) are uncovering the molecular mechanisms that support and regulate metabolic suppression (recent reviews : Storey & Storey, 1990; Guppy, Fuery & Flanigan, 1994 ; Hand & Hardewig, 1996 ; Hochachka et al., 1996 ; Storey, 1996, 2001, 2002 a ; Brooks & Storey, 1997; Guppy & Withers, 1999; Bickler, Donohoe & Buck, 2001 ; Denlinger, 2001; Hochachka & Lutz, 2001). Principles of biochemical regulation are emerging that are widely conserved across phylogenetic lines and show the unifying elements of metabolic arrest. The present review focuses on recent advances in understanding the role of differential gene expression in metabolic depression and the regulatory controls on transcription and translation that allow these energy-expensive processes to be suppressed when organisms enter a hypometabolic state. However, first we will briefly review some of the earlier work on the biochemical regulation of metabolic rate depression, highlighting in particular the central role of reversible protein phosphorylation in coordinating the suppression of many metabolic loci. It should be noted that the research considered herein derives from laboratory experimentation using animals induced to enter a hypometabolic state by the acute imposition of stress (e.g. exposure to a nitrogen gas atmosphere for studies of anoxia tolerance in marine snails). The use of controlled experimental conditions allows the Metabolic rate depression in animals biochemist to identify definitive and typically maximal responses to an imposed stress. However, in nature, the stress conditions that initiate hypometabolism may be much more variable (e.g. less severe, shorter duration, build up slowly over a long period, or be mixed with other stresses) and where possible, biochemical responses identified from laboratory studies should also be assessed in animals sampled from field conditions. II. COORDINATE REGULATION OF METABOLIC RATE DEPRESSION In recent years, a major focus of research in hypometabolism has been the molecular mechanisms that regulate metabolic rate depression. To maintain homeostasis in any cell, the rate of ATP utilization by the myriad of ATP-consuming processes must match the rate of ATP production by central pathways of fuel catabolism. All cells have mechanisms for increasing ATP production when energy demand is high and for scaling back production when energy demand declines. Conversely, situations that limit ATP production will quickly affect the rates of ATP-utilizing processes. Indeed, studies have shown that a hierarchy of sensitivity to ATP supply exists, the pathways of macromolecular biosynthesis (such as protein synthesis, RNA/DNA synthesis) being more sensitive to ATP availability than various other activities such as transmembrane ion pumping (Buttgereit & Brand, 1995). When ATP supply is compromised in intolerant systems, the ability to compensate or adjust to this energy stress is often very limited and cells/organisms quickly succumb to injury or death. The highly injurious effects of hypoxia or hypothermia on the core organs of most mammals offer prime examples of the metabolic damage caused when energy supplies fail (Hochachka, 1986). In particular, ATP limitation has a major effect on one of the most energyexpensive functions of cells, the maintenance of membrane potential difference. ATP limitation causes a rapid imbalance in the opposing rates of ion transport across membranes by ATP-dependent ion pumps versus ATPindependent ion channels so that membrane potential difference is quickly dissipated with multiple negative consequences (Perez-Pinzon et al., 1992 ; Hochachka & Lutz, 2001). It is obvious, then, that the transition from an active state to a hypometabolic or dormant state where metabolic rate (a measure of ATP turnover) may be as little as 1–5% of the former resting metabolic rate of the active organism must require a rebalancing of the rates of both ATPproducing and ATP-utilizing reactions. Metabolic controls must be utilized to coordinate a net suppression of all metabolic functions to achieve a new net rate of ATP turnover that can be sustained over the long-term by the ‘ onboard ’ fuel reserves of the organism. The question is how is this achieved ? Energy savings during hypometabolism come from several sources. Hypometabolism typically goes hand-in-hand with a lack of voluntary muscle movements so the energy costs of skeletal muscle work are nominal. Heart beat 209 and respiration rate are greatly reduced, breathing often becomes intermittent, and kidney filtration rate is reduced. Organisms do not eat so the energetic costs of digestion, nutrient absorption, and peristalsis are eliminated. A substantial part of total energy savings comes from the suppression of these physiological activities. Metabolic rate is also influenced by factors including the partial pressures of O2 and CO2, pH and temperature. Changes in these extrinsic parameters during hypometabolism can account for a portion of the metabolic inhibition (Barnhart & McMahon, 1988 ; Geiser, 1988; Pedler et al., 1996 ; Guppy et al., 2000). Major energy savings in the hypometabolic state come from the specific inhibition of the rates of multiple cellular processes providing an intrinsic component to metabolic arrest. For example, mitochondria isolated from tissues of torpid ground squirrels (Spermophilus sp.) show rates of state 3 respiration that are 30–66 % lower than the rates in euthermic animals (summer active or interbout arousal) yet this stable inhibition is rapidly reversed upon arousal (Fedotcheva et al., 1985; Gehnrich & Aprille, 1988 ; Martin et al., 1999). Whole cells and tissues from hypometabolic animals can also retain an intrinsic metabolic depression when assessed in vitro. The metabolic rate of hepatopancreas cells from estivating land snails (Helix aspersa) was only onethird to one-half that of controls (Bishop & Brand, 2000; Guppy et al., 2000) and the metabolic rate of liver slices from estivating frogs (Neobatrachus centralis) was 45% of that for slices from control frogs (Fuery et al., 1998). The suppression of multiple individual metabolic processes must be coordinated to achieve a net suppression that balances the rates of ATP-producing and ATP-consuming processes at a new lower net rate of ATP turnover. Rates of substrate utilization are always strongly suppressed in hypometabolic states in order to extend greatly the time that the internal fuel reserves can sustain survival. ATP-consuming processes must similarly be suppressed. The concept of ‘channel arrest’ was one of the first ideas to be explored (Perez-Pinzon et al., 1992). If the rate of ATP production is reduced in the hypometabolic state, then the rate of ATP expenditure by ATP-dependent ion pumps must also be reduced and, to maintain homeostasis, the opposing facilitated diffusion of ions through ion channels must also be suppressed. Channel arrest predicts a coordinated suppression of membrane channels and pumps in both plasma and organelle membranes and considerable evidence for the mechanism has accumulated (Bickler et al., 2001; Hochachka & Lutz, 2001). In neurons of anoxia-tolerant turtles (Pseudemys scripta), the main energy-saving mechanism might be the down regulation of firing rates or synaptic transmission involving localized channel suppression and termed ‘ spike arrest ’ by Sick et al. (1993). Other ‘opposing ’ functions should also be coordinately suppressed during entry into the hypometabolic state such as the rates of synthesis versus degradation of macromolecules including DNA, mRNA, proteins and membrane phospholipids. All rate processes should be slowed so that all macromolecules experience significant life extension (perhaps 10-fold or more) and cells remain fully competent to return rapidly to the active state with all metabolic functions intact even after very long periods of dormancy. 210 Priorities for energy expenditure are also re-arranged during hypometabolism to sustain critical functions at the expense of others (Hochachka et al., 1996). This principle was illustrated in studies using isolated hepatocytes from anoxia-tolerant turtles (Chrysemys picta bellii ). Incubation under anoxic conditions resulted in a rapid 94 % decrease in ATP turnover and a reorganization of the proportion of ATP turnover devoted to five main ATP-consuming processes in liver cells : Na+,K+-ATPase activity, protein synthesis, protein degradation, gluconeogenesis, and urea synthesis. Respectively, these consumed 28, 36, 17, 17 and 3 % of ATP turnover in normoxic cells (Hochachka et al., 1996). However, even though the activity of Na+,K+-ATPase decreased by 75 % in anoxic hepatocytes (supporting the theory of channel arrest) (Buck & Hochachka, 1993), the other energy-consuming processes were even more strongly suppressed (e.g. gluconeogenesis was undetectable). As a result the proportion of cellular ATP turnover supporting Na+K+-ATPase activity actually rose to nearly 75% in anoxic cells. However, although multiple metabolic processes are virtually shut down in the hypometabolic state, they must be retained in readiness so that normal metabolic function can be rapidly restored during arousal. Thus, hypometabolism is not generally associated with a major loss of metabolic capacity but instead reversible controls are applied coordinately to suppress the rates of all metabolic processes while leaving intact the potential to return rapidly to the normal state. (1 ) Reversible phosphorylation regulation of metabolic enzymes Much recent work has been aimed at understanding the molecular mechanisms that provide reversible metabolic suppression and coordinate the transitions to and from the hypometabolic state. The regulation of fuel catabolism in hypometabolism was the first area to receive attention, and studies by our laboratory and others showed the repeated occurrence across wide phylogenetic lines of one mechanism that had powerful regulatory control over key enzymes of fuel catabolism. This is reversible protein phosphorylation controlled by the actions of protein kinases and protein phosphatases. The importance of this mechanism was first demonstrated in the regulation of pyruvate kinase (PK) in anoxia-tolerant marine molluscs (reviewed in Storey & Storey, 1990 ; Storey, 1993). Anoxia-induced phosphorylation of PK caused a strong decrease in enzyme activity and major changes in kinetic properties that virtually shut off the enzyme during anaerobiosis. This facilitates a rerouting of carbohydrate flux that directs phosphoenolpyruvate away from PK and instead into the phosphoenolpyruvate carboxykinase reaction and onwards into the reactions of succinate synthesis. Subsequently, the suppression of multiple enzymes of anaerobic glycolysis has been shown to be coordinated by this same reversible phosphorylation mechanism in various species of marine molluscs and other invertebrates and, furthermore, the same mechanisms regulate the suppression of carbohydrate catabolism during aestivation in land snails (Storey, 1993; Brooks & Storey, 1997). Kenneth B. Storey and Janet M. Storey Reversible phosphorylation control over enzymes of fuel metabolism is now recognized as one of the major principles of metabolic regulation in hypometabolism and is known to contribute to metabolic suppression and the reorganization of fuel metabolism in anoxia-tolerant animals, estivating anurans and molluscs, and hibernating mammals (for reviews see Storey, 1996, 1997, 2002 a). For example, inhibition of carbohydrate catabolism during mammalian hibernation (facilitating a switch to lipid oxidation) is aided by a strong phosphorylation-mediated inhibition of pyruvate dehydrogenase (PDH), the enzyme that gates carbohydrate entry into the tricarboxylic acid cycle. During hibernation, the amount of PDH present in the dephosphorylated active a form drops precipitously (Fig. 1 A) so that the percentage of the enzyme that is active falls from 60–80 % in euthermia to less than 5% in hibernation (Brooks & Storey, 1992 a ; Storey, 1997). PDH activity is similarly suppressed in estivating snails (Brooks & Storey, 1992b). The application of metabolic control analysis to isolated hepatopancreas cells from control versus estivating Helix aspersa showed that at least 75 % of the reduction in mitochondrial respiration in aestivation was due to primary changes in the kinetics of substrate oxidation, most probably the result of reduced activities of substratesupplying enzymes (e.g. PDH and others) or enzymes of mitochondrial oxidative metabolism (e.g. citrate synthase and cytochrome c oxidase both decreased in estivating H. aspersa and other land snails) (Bishop, St.-Pierre & Brand, 2000). Reversible phosphorylation control over selected enzymes is a key element in this regulation. ( 2) Reversible phosphorylation regulation of membrane ion channels and receptors Given the role of reversible phosphorylation in the control of fuel catabolism in hypometabolism, and therefore in the rate of ATP generation, it seemed likely that this mechanism should also be involved in regulating the myriad of ATPutilizing processes in cells. Recent studies have addressed the control of energy-expensive metabolic processes beginning with the control of membrane ion pumps. These utilize a huge proportion of total cellular energy to pump ions against their concentration gradients ; for example, the Na+,K+ATPase alone is responsible for 5–40 % of total ATP turnover depending on cell type (Claussen, 1986). Recent experimental studies are showing that reversible phosphorylation control of ion pumps is an integral mechanism of natural hypometabolism. For example, in hibernating ground squirrels, Spermophilus lateralis, Na+,K+-ATPase activities were uniformly reduced in most organs to just 40–60 % of the euthermic values (assayed at 25 xC) (Fig. 1 B). Notably, activity was not suppressed in heart, an organ that must continue to perform muscle work through torpor. In vitro studies confirmed that the mechanism of this inhibition was protein phosphorylation. The activity of skeletal muscle Na+,K+-ATPase from euthermic animals was strongly reduced by incubations that stimulated protein kinases whereas activity was restored after alkaline phosphatase treatment (Fig. 1 C) (MacDonald & Storey, 1999). Activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase is Metabolic rate depression in animals 1.4 A Pyruvate dehydrogenase a 1.2 1.0 0.8 0.6 0.4 0.2 0 * * Heart Kidney Activity (units g –1 wet mass) Activity (units g –1 wet mass) 1.6 211 300 250 200 150 B Na+, K+– ATPase * Euthermic Hibernating 100 80 40 * 20 0 12 Activity (units g –1 wet mass) * 60 Muscle Heart C b Kidney Liver Reversible phosphorylation of Na+, K+– ATPase 10 Control ATP + cAMP Alk. P’tase 8 6 a,b 4 2 a 0 Euthermic Hibernating Fig. 1. Effect of hibernation on the activities of (A) the active form of pyruvate dehydrogenase (PDHa) and (B) Na+,K+-ATPase in tissues of ground squirrels (Spermophilus lateralis). (C) Effects of stimulating endogenous protein kinase A (by incubation with 10 mmol lx1 ATP, 10 mmol lx1 MgCl2, 0.3 mmol lx1 cAMP) and subsequent alkaline phosphatase (Alk. P’tase) treatment (10 units) on Na+,K+-ATPase activity in skeletal muscle extracts from euthermic and hibernating ground squirrels. Data are means¡SEM, N=4. *, Significantly different from the corresponding euthermic value, P<0.05. a – Significantly different from the untreated control sample ; b – significantly different from the protein kinase treated sample, P<0.05. From Brooks & Storey (1992a) and MacDonald & Storey (1999). similarly reduced during hibernation ; both a decrease in enzyme amount (per milligram of SR protein) and in enzyme specific activity (per milligram of Ca2+-ATPase protein) occur in hibernator muscle (Malysheva et al., 2001). The reduction in specific activity is probably due to protein phosphorylation. Other proteins involved in SR calcium signaling are also suppressed during hibernation ; the SR calcium-release channel (ryanodine receptor) decreased by 50 % in hibernation and levels of most SR calcium binding proteins (e.g. sarcalumenin, calsequestrin) were 3–4-fold lower in hibernating, compared with summer-active, animals (Malysheva et al., 2001). The control of neuronal activity, including the regulation of ion pumps, ion channels, and membrane receptors, has been extensively studied in brain of anoxia-tolerant freshwater turtles as a model system for exploring the effects of ischemia on the vertebrate brain (recent reviews Bickler et al., 2001 ; Hochachka & Lutz, 2001). Multiple mechanisms of ion channel arrest have been identified with reversible phosphorylation of channels or receptor subunits playing a substantive role (Bickler et al., 2001). Indeed, all voltagegated channels (Na+, Ca2+, K+) are sensitive to phosphorylation by multiple protein kinases and roles for reversible protein phosphorylation in hypoxic suppression are being identified. One of the most complete pictures of reversible phosphorylation control of neuronal function during hypoxia/anoxia is the regulation of the N-methyl-D-aspartatetype glutamate receptor (NMDAR) (Bickler et al., 2001). Several kinases including protein kinase A, protein kinase C and tyrosine kinase phosphorylate NMDARs and increase activity whereas the opposing phosphatases decrease activity. Suppression of NMDAR activity is critical for the survival of turtle neurons in anoxia and inhibition of phosphatase action (via specific phosphatase inhibitors or by flooding with cAMP to stimulate kinases) prevents the normal silencing of NMDARs under anoxia (Bickler et al., 2001). Most recently, the focus of research on metabolic arrest by our laboratory and others has turned to the regulation of Kenneth B. Storey and Janet M. Storey 212 Table 1. Protein synthesis inhibition during hypometabolism : selected examples from hibernation, anoxia tolerance, aestivation and diapause. The rate of protein synthesis in hypometabolism is shown as a percentage of the corresponding control rate Hypometabolic rate of protein synthesis ( % of control) Species Condition Tissue Spermophilus tridecemlineatus Hibernation Littorina littorea Trachemys scripta Anoxia Anoxia Anoxia Carassius carassius Anoxia Helix aspersa Neobatrachus centralis Austrofundulus limnaeus Artemia franciscana Aestivation Aestivation Diapause Anaerobic quiescence Brain Brain Kidney Brown adipose Hepatopancreas Hepatocytes Heart, liver, brain, muscle, others Liver Heart, muscle Hepatopancreas Liver Whole embryo Whole embryo 0.04* 34 15 104 50 8 y0 5 y50 30 33 7 8 Reference Frerichs et al. (1998) Frerichs et al. (1998) Hittel & Storey (2002 b) Hittel & Storey (2002 b) Larade & Storey (2002 c) Land et al. (1993) Fraser et al. (2001) Smith et al. (1996 b) Smith et al. (1996 b) Guppy et al. (2000) Fuery et al. (1998) Podrabsky & Hand (1999, 2000) Hofmann & Hand (1994) * Compares in vivo protein synthesis rates at euthermic (37 xC) versus hibernating (7.5 xC) body temperatures. Other values for hibernators are for in vitro assays that compare tissue extracts from euthermic and hibernating animals at a constant temperature. other energy-expensive cellular processes (e.g. transcription and translation) and to the role that gene expression plays in producing proteins with specific functions in hypometabolism. The remainder of this review focuses primarily on new studies in these two areas and on our studies with two animal systems in particular – hibernating mammals (ground squirrels of the genus Spermophilus) and anoxia-tolerant marine snails (the periwinkle, Littorina littorea). The new data demonstrate striking parallels in principles and mechanisms of metabolic control that are involved in hypometabolism in both systems. III. METABOLIC ARREST AND SUPPRESSION OF PROTEIN SYNTHESIS Protein synthesis is an energy-expensive process, requiring about five ATP equivalents per peptide bond formed and consuming a substantial portion of the total ATP turnover of all cells and organs (e.g. 36 % in normoxic turtle hepatocytes ; Hochachka et al., 1996). Protein synthesis is well known to be sensitive to the availability of energy and amino acids and the rate of protein synthesis is suppressed during starvation and during severe hypoxia in oxygen-sensitive systems (Casey et al., 2002; DeGracia et al., 2002 ; Mordier et al., 2002). Thus, it is reasonable to predict that protein synthesis would be strongly suppressed in order to save energy during hypometabolism. Indeed, recent studies with several systems confirm this ; strong reductions in the rate of protein synthesis occur as part of hibernation, anoxia tolerance, aestivation and diapause (Table 1) ( Joplin & Denlinger, 1989 ; Hofmann & Hand, 1992b ; Land, Buck & Hochachka, 1993 ; Fuery et al., 1998 ; Hand, 1998; Podrabsky & Hand, 2000; Fraser et al., 2001 ; Larade & Storey, 2002c). For example, in hibernating thirteen-lined ground squirrels (S. tridecemlineatus), the rate of 14C-leucine incorporation into protein in vivo in brain of torpid animals was only 0.04% of the mean value in active squirrels (Frerichs et al., 1998). Part of this rate suppression is due to the body temperature differences between the two states (37 xC for euthermia versus approximately 7.5 xC for torpor) but when protein synthesis by brain extracts was assessed in vitro at 37 xC (eliminating the extrinsic effect of temperature), the intrinsic rate of synthesis in extracts from hibernator brain was still just 34 % of the euthermic value (Frerichs et al., 1998). Similarly, the rate of 3H-leucine incorporation into protein in S. tridecemlineatus kidney extracts in vitro was just 15% of the euthermic value but, interestingly, brown adipose tissue showed no reduction in protein biosynthesis during torpor (Hittel & Storey, 2002b). In addition to an overall suppression of protein synthesis, the proportion of cellular ATP turnover devoted to protein synthesis drops in hypometabolism. In anoxic turtle hepatocytes, for example, ATP use by protein synthesis fell from 24.4% of total ATP turnover under normoxia to just 1.6 % in anoxia (Land et al., 1993). In killifish embryos, the proportion dropped from 36% in normal developing embryos to negligible when embryos entered diapause (Podrabsky & Hand, 2000). Furthermore, suppression of protein synthesis can be a rapid event ; the rate of 3H-leucine incorporation into protein in hepatopancreas extracts from Littorina littorea was reduced by 50 % within just 30 min when snails were transferred from aerobic to anoxic conditions and this value was sustained over 48 h of anoxia exposure (Larade & Storey, 2002 c). This rapid suppression suggests that protein synthesis inhibition is a pro-active response by cells that is an integral part of metabolic arrest rather than a reactive response to ATP limitation. Indeed, ATP content and energy charge did not change significantly over the course of 72 h of anoxia exposure in L. littorea tissues (Churchill & Storey, 1996). Metabolic rate depression in animals Two factors may contribute to the inhibition of protein synthesis during hypometabolism : (1) mRNA substrate availability, and (2) specific inhibition of the ribosomal translational machinery. Substrate availability is always a factor in any metabolic process but there appears to be little, if any, change in global mRNA levels in most hypometabolic systems. In hibernator tissues, for example, neither total RNA nor mRNA levels changed and transcript levels of four constitutive genes did not fall during hibernation, implicating no net loss of the mRNA for constitutively active genes during torpor (Frerichs et al., 1998 ; O’Hara et al., 1999 ; Knight et al., 2000). Evidence from cDNA array screening of hundreds of genes in multiple tissues of ground squirrels and bats also indicates that transcript levels of most genes are unaffected during hibernation (Eddy & Storey, 2001 ; Hittel & Storey, 2001). In anoxia-tolerant turtles, total RNA levels were unaffected in all organs and poly(A)+ RNA levels remained constant in four organs over 16 h of submergence anoxia at 15 xC but rose by 30% in white skeletal muscle (Douglas et al., 1994). Studies with crucian carp (Carassius carassius) suggested that total RNA synthesis (of which the largest component is rRNA) is reduced in brain but increased in heart and liver during anoxia (Smith et al., 1999 a) ; the implications for mRNA content are not clear. Translatable RNA levels did not change in Artemia franciscana during anoxia exposure (Hand, 1998). Furthermore, mRNA appears to remain intact during hypometabolism ; analysis of various specific gene transcripts via Northern blotting failed to detect reductions in transcript sizes during hypometabolism (Cai & Storey, 1996 ; Fahlman, Storey & Storey, 2000 ; Knight et al., 2000 ; Hittel & Storey, 2001, 2002 a, b ; Larade, Nimigan & Storey, 2001; Larade & Storey, 2002 b) and the distribution of poly(A) tail lengths showed no general shortening of tails in torpid ground squirrels as would be expected if transcripts were degrading during hibernation (Knight et al., 2000). Overall, then, global mRNA substrate availability does not appear to be a factor in protein synthesis inhibition in hypometabolism. Control of protein synthesis is vested instead in strong regulation of translation by the ribosomes. Two mechanisms that contribute to the arrest of protein synthesis have received experimental attention : (1) reversible phosphorylation control of the translational machinery, and (2) the state of ribosome assembly. (1 ) Control of translation by reversible protein phosphorylation In brain of hibernating ground squirrels both translation initiation and polypeptide elongation are inhibited (Frerichs et al., 1998). Inhibition of both was traced to the regulation of specific functional proteins in ribosomes. The inhibition of translation initiation was linked with the eukaryotic initiation factor 2 (eIF2) which introduces initiator methionyl-tRNA into the 40S ribosomal subunit (Fig. 2). The mechanism involved is phosphorylation of the alpha-subunit of eIF2 (eIF2a). This mechanism is widely known as a means of inhibiting eukaryotic protein synthesis and even small amounts of phospho-eIF2a significantly inhibit global protein synthesis by blocking the initiation of nascent 213 polypeptides (Rhoads, 1993 ; Mikulits et al., 2000). For example, in rat brain homogenates obtained after 90 min reperfusion following 10 min cardiac arrest, protein synthesis was reduced by 85 % and this was associated with approximately 23 % of eIF2a in the phosphorylated state compared with approximately 1 % under control conditions (DeGracia et al., 2002). Phosphorylated eIF2a acts as a dominant inhibitor of the guanine nucleotide exchange factor eIF2B and prevents the recycling of eIF2a between successive rounds of peptide synthesis (Clemens, 2001 ; DeGracia et al., 2002). Conditions known to increase eIF2a phosphorylation include virus infection, heat shock, iron deficiency, nutrient deprivation, changes in intracellular [Ca2+], induction of apoptosis, the unfolded protein response, hypoxia/anoxia and postischemic reperfusion (Munoz et al., 2000 ; Althausen et al., 2001; Clemens, 2001; Martin de la Vega et al., 2001; Casey et al., 2002 ; DeGracia et al., 2002). Analysis of eIF2a regulation uses two kinds of antibodies – one that detects eIF2a protein in general and one that is specific for the peptide containing the phosphorylated residue. This analysis has consistently shown a strong increase in the amount of phospho-eIF2a in hypometabolic states. For example, phospho-eIF2a content increased markedly in kidney of hibernating squirrels but with no change in total eIF2a protein (Hittel & Storey, 2002 b). In brain of euthermic ground squirrels phospho-eIF2a content was less than 2% of the total but rose to approximately 13% during hibernation (Frerichs et al., 1998). The same mechanism occurs during anoxia in the marine snail, L. littorea. Total eIF-2a content in hepatopancreas was unaffected over a cycle of anoxia and aerobic recovery but the amount of phospho-eIF2a rose approximately 15-fold in anoxic animals, compared with aerobic controls (Fig. 3) (Larade & Storey, 2002c). However, when oxygen was reintroduced, phospho-eIF2a fell to control levels or below within 1 h. Although the focus to date on the suppression of translation initiation in hypometabolism has been on eIF2a, other regulatory factors may be involved and these await study in hypometabolic systems. For example, the eukaryotic initiation factor 5 (eIF5), which acts as a GTPase-activating protein to promote GTP hydrolysis within the 40S initiation complex (consisting of 40S*eIF3*AUG*Met-tRNA(f)*eIF2* GTP) (Fig. 2), is also regulated by reversible protein phosphorylation (Majumdar et al., 2002). Eukaryotic initiation factor 4E binding protein (4E-BP1) is also regulated in this manner (Fig. 2) and was significantly dephosphorylated during ischemia in rat brain (at the same time as eIF2a was phosphorylated) (Martin de la Vega et al., 2001). Dephosphorylation allows the 4E-BP1 to bind to and inhibit eIF4. Another subunit of this initiation factor, eIF4G, shows proteolytic fragmentation after ischemia/reperfusion in rat brain. Fragmentation changes the types of mRNAs that can be translated because intact eIF4G is needed to allow eIF4E-bound m7G-capped mRNAs (the vast majority of cellular mRNAs) to bind to the small ribosomal subunit (DeGracia et al., 2002). Without intact eIF4G, message selection changes dramatically to favour only those messages that contain an internal ribosome entry site (IRES) (Gingras, Raught & Sonenbert, 1999). In mammals, many of the messages that contain an IRES code for proteins 214 Kenneth B. Storey and Janet M. Storey Fig. 2. Mechanism of translation initiation focusing on the control of eukaryotic initiation factors 2 (eIF2) and 4 (eIF4) via reversible protein phosphorylation. Hexagonal figures show initiation factors, indicated with numbers ; ovals show ribosomal subunits and eIF Metabolic rate depression in animals involved in apoptosis and recent work suggests that the protein synthesis inhibition response, characterized by eIF2a phosphorylation and eIFG fragmentation, is typical of the initiation of apoptosis in response to various stresses (summarized by DeGracia et al., 2002). Amino acid starvation also inhibits protein synthesis through these same mechanisms and leads to up-regulation of selected gene transcripts containing an IRES despite the general inhibition of the cap-dependent translational apparatus during starvation (Fernandez et al., 2001 ; Mordier et al., 2002). Hypometabolism is, of course, a form of starvation ; organisms stop eating and switch to a dependence on internal fuel reserves. It is possible, then, that the protein synthesis inhibition response and/or other selected adjustments for hypometabolism grew out of the pre-existing mechanisms that regulate suppression of non-essential processes either (a) during starvation or (b) in response to ischemic stress. Modified controls or an additional layer of control could be added to avoid or reverse signals that would in other organisms lead to apoptosis. Furthermore, it is seems probable that message selection (via an IRES or other mechanism) represents the key to the specific up-regulation of selected genes during natural hypometabolism. Of note in this regard is a key new study that demonstrates the presence of an IRES in the mRNA transcript of hypoxia-inducible factor-1a (HIF-1a) (Lang, Kappel & Goodall, 2002). HIF-1 mediates a variety of gene upregulation responses to hypoxia in oxygen-sensitive organisms to produce proteins that either increase anaerobic ATP production such as glycolytic enzymes and glucose transporters or enhance oxygen delivery such as erythropoietin and vascular endothelial growth factor (VEGF) (Bunn & Poyton, 1996). HIF-1 activity is primarily regulated via availability of the HIF-1a subunit which is expressed constitutively but rapidly degraded ; by contrast, HIF-1b subunit levels are always substantial. When oxygen is low, degradation is inhibited and HIF-1a content increases, dimerizes with HIF-1b and translocates to the nucleus to activate gene expression. However, to be effective in hypoxia signaling, the translation of HIF-1a itself must continue under hypoxia. Using polysome profile analysis, Lang et al. (2002) demonstrated that HIF-1a mRNA remained in the polysome fraction during hypoxia (despite an overall 50% decrease in protein synthesis in hypoxic cells). The long and GC-rich 5k-untranslated region (UTR) of HIF-1a suggested the presence of an IRES and this was confirmed with a series of transfection studies using luciferase downstream reporters (Lang et al., 2002). The presence of this IRES allows HIF-1a translation to be maintained under hypoxic conditions that are inhibitory to cap-dependent translation. Interestingly, VEGF also contains an IRES (Miller et al., 1998). As will be discussed in a later section, new research with both mammalian hibernators and anoxia-tolerant snails shows that 215 a number of genes that are up-regulated during hypometabolism are distributed very differently across a polysome/ monosome gradient than are constitutively expressed genes and it can be proposed that the mRNA transcripts of these genes may contain an IRES in the 5kUTR that allow their translation during hypometabolism. The upstream regulators of the initiation factors also need study. Mammalian cells have four eIF2 kinases, each activated by a distinct stress. Heme-regulated inhibitor (HRI) is activated by hemin deprivation, double-stranded RNAdependent kinase (PKR) is activated by double-stranded RNA, PKR-like endoplasmic reticulum kinase (PERK) is activated by unfolded proteins and ischemia/reperfusion, and the general amino acid control eIF2 protein kinase (GCN2) is activated by serum or amino acid starvation (Kumar et al., 2001). Identification of the protein kinase(s) that mediates the suppression of eIF2a during hypometabolism will be a major advance since this kinase will likely also regulate various other downstream targets. Furthermore, Harding et al. (2000) showed that whereas the eIF2a kinases, PERK and GCN2, repress translation of most mRNAs by targeting eIF2a, they also selectively increase translation of the transcription factor ATF4 (activating transcription factor) which in turn increases transcription of selected genes. This suggests a method by which a single signal, in this case a signal acting via PERK, can have both generalized (inhibition of protein synthesis) and specific (up-regulation of selected genes) actions and may provide a model for understanding similar situations of selective gene up-regulation during hypometabolism. Regulation of eIF2a could also come from control over the dephosphorylation of the phosphoprotein. Both protein phosphatase (PP) 1 and 2A can dephosphorylate phosphoeIF2a and PP-1 appears to have this role in vivo (Redpath & Proud, 1990). However, studies to date have given mixed results as to whether inhibitory control of phosphatases is a factor in phospho-eIF2a accumulation during ischemia/ reperfusion (Munoz et al., 2000 ; DeGracia et al., 2002). Interestingly, analysis of the effects of natural hypometabolism (in estivating or anoxia-tolerant organisms) on protein phosphatase activities have shown that the general response is a reduction in PP-1 and PP-2A activities in tissues (Mehrani & Storey, 1995 ; Cowan et al., 2000 ; Cowan & Storey, 2001) consistent with the general metabolic rate depression and with the dominant role of protein kinases in suppressing metabolic functions during entry into hypometabolism. Protein translation is also regulated at the level of polypeptide elongation and reversible phosphorylation control over specific elongation factors is again the mechanism. For example, in both mouse and rat models of brain ischemia, phosphorylation of the eukaryotic elongation factor-2 (eEF-2) increased during ischemia and decreased again inhibitor proteins. The figure shows the order of assembly of various eIFs onto the 40S ribosomal subunit including eIF2 which carries the initiator methionyl-tRNA and the eIF4 family that assemble on the m7G-capped mRNA, with its start codon (AUG), and introduce it to the 40S subunit. Shaded boxes show (a) the recycling of eIF2 and inhibitory control of this factor via phosphorylation of its a-subunit which stabilizes the interaction of eIF2-GDP with its binding protein, eIF2B, and (b) inhibitory control of eIF4E via interaction with its binding protein (4E-BP) which is released when 4E-BP is phosphorylated. Kenneth B. Storey and Janet M. Storey 216 Protein ? Ribosomal Protein L26 Gene 60S Normoxia Anoxia Recovery P elF-2a A Protein 40S Met-tRi elF-2a-P elF-2 GTP Fig. 3. Analysis of ribosomal components during anoxia exposure in Littorina littorea. The top left panel shows the increased expression of ribosomal protein L26 mRNA in hepatopancreas over the course of anoxia exposure (12, 24, 96 and 120 h anoxia) and aerobic recovery (1 h recovery after 120 h anoxia). The bottom left panel shows changes in the phosphorylation of the a subunit of eukaryotic initiation factor 2 (eIF-2a) after 24 h anoxia or 1 h recovery after anoxia. Both L26 and eIF-2 play roles in efficient translation, and the right panel shows the location of both components on the intact ribosome. L26 is located at the subunit interface and functions during the peptide transfer from the A (aminoacyl) site to the P (peptidyl) site, whereas eIF-2, which is involved in initiation, is associated with the A site. Modified from Larade et al. (2001) and Larade & Storey (2002 c). during reperfusion (Althausen et al., 2001; Martin de la Vega et al., 2001). To date, control of elongation factors in natural hypometabolism has only been studied in hibernating ground squirrels. Frerichs et al. (1998) demonstrated that mean transit times for polypeptide elongation by ribosomes were three-fold longer in extracts from brain of hibernating ground squirrels, compared with euthermic animals. Subsequently, elevated amounts of phospho-eEF-2 were found in brain and liver of hibernating ground squirrels, compared with euthermic controls (Chen et al., 2001). Regulation was due to both an approximately 50 % higher activity of eEF-2 kinase in hibernator tissues and a 20–30 % decrease in PP-2A activity (which opposes eEF-2 kinase). This latter was the result of a 50–60% increase in levels of the specific inhibitor of PP2A, I2PP2A (Chen et al., 2001). Interestingly, recent work by Ryazanov (2002) has cloned and sequenced eEF-2 kinase and found that it represents a new family of kinases. Sequence comparison has identified two other family members, so-called channel-kinases, that represent a novel type of signaling molecule – a protein kinase fused to an ion channel. Thus, another huge field of protein kinase mediated signal transduction is now open for exploration. For the field of hypometabolism, this new family of kinases offers an intriguing potential way of coordinating both channel arrest and protein synthesis arrest. The involvement of protein phosphorylation mechanisms in the control of both translation initiation and peptide elongation further emphasizes the key role that this regulatory mechanism plays in multiple aspects of metabolic suppression and links the suppression of protein synthesis with the control of multiple other metabolic functions (e.g. enzymes of fuel metabolism, ion-motive ATPases) discussed previously. The important principle here is that multiple cell functions are controlled via a single regulatory mechanism (reversible protein phosphorylation) to achieve a coordinated suppression of metabolic rate. ( 2) Ribosome aggregation state The activity state of the protein-synthesizing machinery in a cell/tissue can generally be inferred from the state of ribosomal assembly. Active translation occurs on polysomes (aggregates of ribosomes moving along a strand of mRNA) whereas monosomes are translationally silent. Metabolic rate depression in animals 0.06 217 A 0.05 B P M P A 254 0.04 M 0.03 0.02 0.01 0 Relative band intensity 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 3000 2000 1000 0.06 A 254 D C 0.05 P P M 0.04 0.03 M 0.02 0.01 0 Relative band intensity 1 2 3 4 5 6 7 8 9 10 1 15% 30% 2 3 4 5 6 7 8 9 10 3000 2000 1000 30% Linear sucrose gradient Linear sucrose gradient 15% Fig. 4. Polysome profiles of Littorina littorea hepatopancreas extracts. Tissue samples were gently homogenized in buffer containing 300 mmol lx1 sucrose and centrifuged at 13 000 g to pellet mitochondria. Supernatants were removed and then centrifuged at 40 000 g on 15–30 % continuous sucrose density gradients and then fractions were collected. The absorbance at 254 nm (A254) (upper panel of each triplet) shows the relative amount of total RNA in each fraction. Pairs of fractions were then pooled, separated by agarose gel electrophoresis and stained with ethidium bromide (middle panel) or blotted onto nitrocellulose for Northern blotting with 32P-labeled a-tubulin probe ; the scanned intensity of tubulin bands is shown in the lower panel. (A) Aerobic, (B) 24 h anoxia, (C) 72 h anoxia, (D) 72 h anoxia followed by 3 h aerobic recovery. Polysome region [P] ; monosome peak [M]. Plots are representative of N=3 trials per condition. Modified from Larade & Storey (2002 c). Hence, an effective way to gauge the effects of a stress on cellular translational activity is to analyze changes in the proportions of polysomes versus monosomes. A polysome profile is generated by separating ribosomes on a sucrose gradient (Surks & Berkowitz, 1971). Polysomes appear in the denser fractions (e.g. see fractions 3–6 in Fig. 4) with the lighter weight monosomes and messenger ribonuclear proteins (mRNP) in the less dense fractions (fractions 7 and higher). Ribosome presence throughout the gradient is quantified by detecting ribosomal RNA in one of several ways : measurement of absorbance at 254 nm, ethidium bromide staining, or Northern blotting with a 32P-labelled probe specific for 18S rRNA. Stresses that compromise cellular energy or amino acid availability lead to dissociation of polysomes and an increase in the number of free translationally silent monosomes. For 218 example, hypoxia/anoxia stress, starvation and diabetes have this effect (Surks & Berkowitz, 1971 ; Metter & Yanagihara, 1979 ; Harmon, Proud & Pain, 1984). Studies with several systems have now shown that polysome disaggregation is a component of natural hypometabolism. For example, in response to anoxia exposure A. franciscana embryos show a decrease in polysome content as one part of a 92 % suppression of overall metabolic rate (Hofmann & Hand, 1992). Polysome disaggregation also occurs in mammalian hibernation and during anaerobiosis in marine molluscs (Frerichs et al., 1998; Knight et al., 2000 ; Hittel & Storey, 2002b ; Larade & Storey, 2002 c). Fig. 4 illustrates this principle showing the effects of anoxia exposure on ribosome distribution patterns in extracts of L. littorea hepatopancreas. Under normoxic conditions, a high proportion of ribosomes was present in the high-density polysome fractions as confirmed by both absorbance measurements at 254 nm and ethidium bromide staining for RNA. The mRNA for a constitutively expressed gene, tubulin, was also mainly in the polysome fraction during normoxia (Fig. 4 A). This is consistent with a state of active translation under aerobic conditions. After 24 h of anoxia exposure, however, the peak of ribosomal RNA began to shift towards a lower density indicating disagreggation of polysomes into monosomes (Fig. 4B) and after 72 h of anoxia there was little evidence of polysomes remaining in hepatopancreas extracts (Fig. 4 C). Tubulin mRNA was also sequestered into the monosome peak during anoxia which is consistent with the idea that the mRNA pool is largely maintained in the hypometabolic state but is not translated. Within 3 h of the shift back to an oxygenated atmosphere, however, all indicators showed a major reassembly of polysomes and a return of mRNA for constitutively active genes into the polysome pool (Fig. 4D). Comparable studies with several tissues (brain, liver, kidney) of ground squirrels showed the same principle of polysome disaggregation during hibernation and a shift of mRNA for constitutively active genes into the monosome fraction (Frerichs et al., 1998; Knight et al., 2000 ; Hittel & Storey, 2002b). For example, in euthermic kidney extracts there was a distinct distribution of the mRNA for the constitutively expressed gene, cytochrome c oxidase subunit 4 (Cox4 ), between the heavy polyribosome and the monosome/mRNP fractions. However, in hibernator extracts, Cox4 mRNA transcripts were strongly shifted into the monosome fraction (Hittel & Storey, 2002 b). Knight et al. (2000) found a similar shift of the mRNA for another constitutive enzyme (glyceraldehyde-3-phosphate dehydrogenase) into the monosome fraction during hibernation with mRNA returning to polysome fractions during arousal. Furthermore, by sampling ground squirrels at different body temperatures during entry into and arousal from hibernation, van Breukelen & Martin (2001) showed a temperature dependence of polysome disaggregation in liver. The distribution of the mRNA for actin, a constitutively expressed gene, was monitored during cooling, and 18 xC was found to be the critical temperature where polysome disaggregation began, as judged by a large increase in actin mRNA and rRNA in the monosome fraction. Similarly, reaggregation of polysomes also occurred at this temperature during arousal from hibernation. Whether Kenneth B. Storey and Janet M. Storey this effect of temperature on ribosome aggregation state is a passive influence of temperature or results from regulation of one of the initiation factors remains to be seen. Several well-known examples of temperature-dependent metabolic switches are in fact mediated by the differential effects of temperature on the activities of selected protein kinases and phosphatases. The activation of cryoprotectant synthesis at 5 xC in cold-hardy insects is a prime example of this, the effect deriving from a specific low temperature inhibition of glycogen phosphorylase phosphatase (PP-1) which then allows phosphorylase kinase activity to dominate and lead to an activation of glycogenolysis (Storey & Storey, 1991). Studies with mammalian systems have linked the suppression of protein synthesis and the phosphorylation of eIF2a during nonlethal disruptions of cellular homeostasis with the assembly of stress granules, ribonuclear protein structures that reversibly bind and protect mRNAs (Kedersha et al., 1999). Storage of mRNAs in such stress granules during hypometabolism would be a useful mechanism that could (a) preserve the valuable pool of untranslated mRNAs until normal conditions were re-established, and (b) provide for a very rapid re-initiation of the translation of key transcripts to provide protein products that are needed immediately during arousal from the hypometabolic state. Prominent protein constituents of stress granules are poly(A)-binding protein 1 (PABP-1) and T-cell intracellular antigen-1 (TIA-1), a self-aggregating RNA-binding protein (Kedersha et al., 1999). To determine whether these proteins played a role in hypometabolism during hibernation, Western blotting was used to assess their presence in the polysome profiles of kidney extracts from euthermic versus hibernating ground squirrels (Hittel & Storey, 2002b). TIA1 was restricted to the monosome/RNP fractions in both situations but a significant redistribution of PABP-1 was seen during hibernation (Fig. 5). In extracts from euthermic animals, PABP-1 was found only in monosome fractions (fractions 8–10) whereas in extracts from hibernating individuals PABP-1 was also detected in fractions 4, 5, and 7, fractions that also contained substantial amounts of Cox4 and Oct2 mRNA. This suggests that a significant amount of the mRNA in these fractions is sequestered into untranslatable pools. Furthermore, the presence of ultrastructural changes in the nuclei of hibernating animals and indirect evidence for the binding of PABP to hibernator mRNAs (Wang & Lee, 1996 ; Knight et al., 2000) strengthens the argument for the presence of stress granules during torpor. If most polysomes break down during hypometabolism and if most mRNA transcripts are sequestered into untranslatable pools, then how are genes that are up-regulated during torpor handled ? Studies with hibernating S. tridecemlineatus are highlighting some interesting variations on the general principle. ( a ) Differential distribution of individual mRNA species Brown adipose tissue (BAT) is a thermogenic organ that is critical to survival of the hibernator because it is a major producer of the heat that is needed to rewarm the animal during arousal. Unlike the situation in other tissues of the Metabolic rate depression in animals 219 A B Relative transcript levels E 2.5 H OCT2 COX4 C Oct2 mRNA 2.0 1.5 1.0 0.5 0 E H D 20 1 2 3 4 5 6 7 8 10 % mRNA E 9 H Oct2 mRNA 15 10 5 E 0 1 2 3 4 5 6 7 8 9 10 Fraction number E kDa 1 PABP-1 81 TIA-1 43 2 3 Euthermic 4 5 6 7 8 9 10 1 2 3 Hibernating 4 5 6 7 8 9 10 Fig. 5. Effect of hibernation on the organic cation transporter 2 (OCT2) and mRNA binding proteins in Spermophilus tridecemlineatus kidney. (A) Western blots showing levels of OCT2 protein compared with a constitutive protein, cytochrome c oxidase subunit 4 (COX4), in euthermic (E) versus hibernating (H) animals. (B) Oct2 mRNA transcript levels. Values are means¡SEM, N=3. (C) Kidney extracts were separated on 0.5–1.5 mol lx1 sucrose gradients drained into 10 fractions (fraction numbers increase with decreasing density), and total RNA in each fraction was run on an agarose gel, blotted onto nylon membranes and Northern blots were hybridized with 32P-dCTP-labeled Oct2 cDNA probe. (D) Blots in C were scanned and mRNA band intensity in each fraction was plotted as a percentage of the total mRNA signal detected. Data points are means for N=3 trials with triangles showing euthermic samples and squares showing hibernating samples. (E) Western blot analysis of poly(A)-binding protein 1 (PABP-1 ; 81 kDa) and T-cell intracellular antigen-1 (TIA-1 ; 43 kDa) protein levels in the polysome profile fractions. Modified from Hittel & Storey (2002b). hibernator, the rate of protein synthesis in BAT was not reduced in hibernation (Table 1) and there was actually an increased abundance of 18S rRNA in the higher density polysome fractions of BAT from hibernating S. tridecemlineatus compared with euthermic animals (Hittel & Storey, 2002 b). The distribution of two mRNA transcripts on the polysome profile was followed, one that is up-regulated during hibernation (the H isoform of fatty acid binding protein ; H-FABP) and one that is constitutively expressed (Cox4 ) (Fig. 6). H-FABP has a critical function in BAT of hibernators in the intracellular transport of fatty acids, the fuel for thermogenesis. Transcript levels of H-FABP increased approximately threefold in BAT during hibernation (Hittel & Storey, 2001) and a high proportion of this message was found associated with the heaviest polysomes (Hittel & Storey, 2002 b). Compared with euthermic controls, this represented a substantial enrichment of the H-FABP transcript in the heavy polysome fraction and correlated with the observed threefold increase in H-FABP protein in BAT from hibernating animals. By contrast, when the distribution of mRNA for the constitutively expressed gene was assessed, most Cox4 mRNA was sequestered into the monosome and mRNP fractions during hibernation and COX4 protein levels remained constant. This indicates that individual transcript species can be treated differently during hibernation and suggests that another level of metabolic control in hibernation is the differential distribution of individual mRNA species between translationally active and inactive ribosomes. The heavy polyribosomes in hibernator BAT appear to contain those mRNAs (such as H-FABP) that are crucial to the hibernation phenotype while mRNA species that are not needed during hibernation are relegated into the translationally silent monosome fractions. Kenneth B. Storey and Janet M. Storey 220 D A 4 5 6 7 8 9 10 % mRNA 1 2 3 E H 1 2 3 4 5 6 7 8 9 10 E % mRNA B E H 40 35 30 25 20 15 10 5 0 20 4 5 6 7 8 9 10 E H F % mRNA 1 2 3 1 2 3 4 5 6 7 8 Fraction number 9 10 G H-FABP mRNA E H H-FABP 15 10 5 0 C 18S rRNA COX4 1 2 3 4 5 6 7 8 Fraction number 9 10 30 Cox4 mRNA 25 20 15 10 5 0 1 2 3 4 5 6 7 8 Fraction number 9 10 Fig. 6. Effect of hibernation on heart-type fatty acid binding protein (H-FABP) compared with a constitutive protein, cytochrome c oxidase subunit 4 (COX4), in brown adipose tissue of Spermophilus tridecemlineatus. (A–C) Northern blots showing the distribution of 18S rRNA transcripts (which tracks the position of the 40S ribosomal subunit), H-FABP mRNA transcripts, and COX4 transcripts in polysome profiles of brown adipose tissue from euthermic (E) versus hibernating (H) squirrels. (D–F) Band intensities plotted as a percentage of the total signal detected. Other information as in Fig. 5. (G) Western blot analysis of H-FABP and COX4 protein levels. Modified from Hittel & Storey (2002b). (b ) Anticipatory up-regulation Another variation on translational control is the concept of anticipatory up-regulation of genes. Transcript levels of some genes are elevated in the hypometabolic state but no increase in the level of the corresponding protein product occurs. Analysis of polysome profiles indicates that this is because the up-regulated mRNA transcripts are sequestered into the monosome fraction in the hypometabolic state. The gene for the organic cation transporter type 2 (Oct2 ) in hibernator kidney represents a case in point (Fig. 5). Organic cation transporters are transmembrane protein pumps that actively absorb and/or excrete endogenous and exogenous organic ions against their concentration gradients (Burckhardt & Wolff, 2000 ; Urakami et al., 2000). OCT2 protein is found primarily in kidney where it is localized in the basolateral membranes of cells lining the proximal tube (outer medulla) of the nephron. During hibernation transcript levels of Oct2 were 2–3-fold higher in kidney compared with controls (assessed by both Northern blots and cDNA arrays) (Hittel & Storey, 2002b). Despite this, Western blots showed that OCT2 protein levels dropped by 66% in hibernator kidney. This dichotomy was explained by the distribution of Oct2 mRNA on the polysome profile. In euthermic extracts, Oct2 mRNA was partitioned between the heaviest polysomes (fractions 1 and 2) and the unbound mRNA or mRNPs (fraction 9) (Fig. 5 D). During hibernation, however, the profile showed both a strong increase in the total amount of Oct2 message (by 2.1-fold) and surprisingly, that the vast majority of this mRNA was localized in fractions 6–9. Thus, although total Oct2 transcript levels clearly increased during hibernation in kidney, most of the transcripts were stored in the translationally silent monosome and mRNP fractions. Hence, hibernation stimulated transcription of Oct2 but not its translation into protein. Why would this be ? One reason may be that OCT2 protein could be particularly sensitive to some form of damage that accrues during hibernation ; for example, the protein may be damaged by oxygen free radicals or by low temperature, leading to its degradation. Another possibility is that part of the process of shutting down kidney function during hibernation may be the suppression of membrane transport functions that are major energy consumers in kidney. Some transporters may be controlled with reversible mechanisms such as protein phosphorylation whereas deactivation of others may be only possible via protein degradation. In either case it could make sense that the gene is up-regulated as animals enter hibernation and its transcripts are stored in the monosome/ mRNP fractions during torpor. That way the transcripts are Metabolic rate depression in animals already present and ready to be translated as soon as arousal begins in order to allow the fastest resumption of kidney functions during the brief hours of the interbout. IV. METABOLIC ARREST AND SUPPRESSION OF PROTEIN DEGRADATION Net protein turnover is governed by the rates of both protein synthesis and protein degradation. Given that protein synthesis is strongly suppressed in hypometabolic states but that, upon arousal, animals show no significant deficit of cellular proteins, it is obvious that the rates of protein degradation must also be strongly suppressed during hypometabolism. Indeed, these two opposing processes are undoubtedly coordinately regulated to achieve a lower net rate of protein turnover. In addition to the ATP savings associated with suppressed protein turnover during hypometabolism, the protein ‘life extension ’ that results would also minimize the amounts of nitrogenous end products that accumulate as by-products of proteolysis and reduce the costs of processing and storing these during torpor. For example, the rate of urea synthesis by anoxic turtle hepatocytes was reduced by 70% compared with controls (Hochachka et al., 1996). Studies aimed at assessing proteolysis in hypometabolic states are considerably fewer and more variable in their methodology than those that have addressed protein synthesis but estimates of protein longevity in hypometabolic states indicate that proteolysis must be suppressed. For example, the half-life of cytochrome oxidase was increased by as much as 77-fold in encysted gastrulae of the brine shrimp, A. franciscana, indicating strong suppression of proteolysis (Anchordoguy & Hand, 1995). Land & Hochachka (1994) assessed proteolysis in both labile and stable protein pools in isolated hepatocytes of anoxia-tolerant painted turtles (C. p. bellii ). Compared with controls, half-lives of the labile protein pool increased by 40 % in anoxic hepatocytes, halflives of stable proteins doubled, and the mean suppression of proteolysis for both pools combined was 36 %. Furthermore, proteolysis in aerobic hepatocytes accounted for 22% of total energy use by hepatocytes but this was suppressed to just 0.7 % under anoxia. Recall that the proportion of ATP turnover devoted to protein synthesis in aerobic versus anoxic turtle hepatocytes was comparable (Land et al., 1993), indicating that rates of protein synthesis and degradation are coordinately regulated. The mechanisms of proteolytic suppression in hypometabolism are just beginning to be identified. Ubiquitindependent proteolysis is one of the major modes of cellular proteolysis; conjugation with a polymer of ubiquitin tags proteins for degradation by the 26S proteasome whereas monoubiquitylated proteins are targeted for endocytosis and degradation in lysosomes (Pickart, 2001). A recent study with ground squirrels showed that the level of ubiquitinconjugated protein rose by 2–3-fold during hibernation, an occurrence that is best explained by an inhibition of proteolysis during torpor, a process that in mammalian cells is highly sensitive to temperature reduction (van Breukelen & 221 Carey, 2002). Other studies with A. franciscana, suggest that the block on proteolysis during hypometabolism is placed at the level of ubiquitin conjugation because the ubiquitin conjugate levels in anoxic embryos fell to just 7% of normoxic values (Anchordoguy & Hand, 1994). The resumption of ubiquitination in embryos during recovery from anoxia was dependent on a rise in ATP levels (and/or reduced AMP concentration) and a re-alkalization of the cytoplasm (Anchordoguy & Hand, 1995). Data from anoxiatolerant turtles might also support control of proteolysis at the level of protein ubiquitination because the activity of the multicatalytic proteinase complex (a key part of the 26S proteosome) did not change in liver of anoxic versus control turtles (Willmore & Storey, 1996). However, there is not yet sufficient information from different animal systems to suggest whether either, both or none of these represent the common mode of proteolysis control in hypometabolic animals nor are the regulatory mechanism(s) of proteolysis suppression known. V. METABOLIC ARREST AND GENE EXPRESSION (1 ) RNA synthesis Protein synthesis has two major energetic costs, the cost of transcription and the cost of translation. As discussed above, the suppression of protein translation in hypometabolic states is a major contributor to energy savings. Is transcription similarly suppressed ? It is estimated that transcription consumes 1–10 % of the energy budget of cells (Rolfe & Brown, 1997). Presumably global transcription rates would also be suppressed in hypometabolism although there has not been much study of this. Work with A. franciscana showed that during quiescence induced by anoxia the global arrest of protein synthesis was accompanied by mRNA levels that remained constant (Hand & Hardewig, 1996). This indicated that (a) protein synthesis inhibition is not caused by message limitation, and (b) the rate of transcription must also be arrested. The latter point was further confirmed using nuclear run-on assays. These showed that the rate of transcript elongation was reduced by 79 and 88% after 4 and 24 h of anoxia exposure, respectively (increasing mRNA half-lives by at least 8.5-fold), yet was fully restored within 1 h when embryos were reoxygenated (van Breukelen, Maier & Hand, 2000). Artificial acidification of aerobic embryos indicated that approximately half of the anoxiainduced suppression of transcription could be accounted for by pH effects on the process. A similar analysis of transcription during hibernation showed an approximately twofold decrease in transcript initiation in liver of hibernating S. lateralis versus interbout aroused animals. This, coupled with Q 10 values of 2–3 for the rate of mRNA transcript elongation in vitro indicated that transcript elongation would be virtually halted in vivo at the low body temperatures typical of hibernation (van Breukelen & Martin, 2002). Application of the nuclear run-on technique to L. littorea hepatopancreas showed a similar suppression of the rate of mRNA transcription during anaerobiosis ; the rate of 222 mRNA elongation under anoxic conditions, measured as 32 P-UTP incorporation into nascent mRNAs by isolated nuclei, dropped to less than one-third of the normoxic rate (Larade & Storey, 2002 a). (2 ) Gene discovery The transition to a hypometabolic state is certainly not the time for a major restructuring of cellular metabolism and, indeed, evidence from a variety of sources indicates that the vast majority of cellular proteins are unaltered when organisms make this transition. Instead, reversible regulatory controls are applied to multiple key loci to provide both a coordinated suppression of net ATP turnover and a readjustment of selected functions to serve the needs of the hypometabolic state. The prominence of reversible controls in hypometabolism means that relatively few changes to the protein composition of cells may be required to support the transitions to and from the hypometabolic state. Indeed, based on the results of various survey studies of stressinduced gene expression (described below), this is proving to be the case. All studies seem to show that changes in gene and protein expression associated with metabolic depression are highly selective and tend to serve specific needs of the hypometabolic state. Analysis of the gene expression changes that support hypometabolism is in its infancy and, to date, the results have been quite spotty and derived from multiple species/tissues and diverse techniques. Common principles that extend across phylogenetic lines have yet to be identified but major advances in the technology for gene screening, as well as emerging technologies for protein screening, are certain to allow important discoveries over the next few years. Various methods of gene discovery have been used to date including cDNA library screening, differential display polymerase chain reaction (PCR) and cDNA array screening. Differential screening of cDNA libraries is especially useful for the detection of novel genes that are specific to the animal or stress under consideration. For example, the anoxia-induced gene, kvn, was discovered this way in L. littorea hepatopancreas, its sequence showing no similarities to any other gene/protein sequences present in international databases (Larade & Storey, 2002 b). Novel genes specific to freezing survival have also been found in freeze-tolerant frogs (Storey, 1999). One new advance in molecular biology will revolutionise gene screening over the next few years: the use of cDNA arrays. These will allow broad-based and comprehensive screening for stress-induced gene expression patterns. Stateof-the-art glass slide microarrays can have up to 31 500 nonredundant cDNAs bound to them (Boer et al., 2001) and offer one-step screening of the responses of broad families of genes (e.g. signal transduction systems, glycolytic enzymes, molecular transporters, transcription factors) to an imposed stress. Array screening is currently having a major impact in medical research allowing, for example, broad screening of gene expression changes during aging, disease, and carcinogenesis (Hal et al., 2000; Boer et al., 2001 ; Helmberg, 2001). We have recently used both nylon macroarrays (Clontech Rat ATLASTM containing cDNAs for 588 genes) Kenneth B. Storey and Janet M. Storey and human 19 000 cDNA microarrays (Ontario Cancer Institute) to screen for hibernation-specific gene expression in ground squirrels and bats (Eddy & Storey, 2001, 2002 ; Hittel & Storey, 2001) and anoxia-induced gene expression in L. littorea (Larade & Storey, 2002 a). Nylon macroarrays were used to examine gene expression in brown adipose tissue of hibernators. These showed that transcript levels of most genes did not change during hibernation but expression of a few (<1 %) was elevated. Prominent among these were isoforms of FABP (Hittel & Storey, 2001). Similar screening of S. tridecemlineatus skeletal muscle showed that several genes that encode components of the small and large ribosomal subunits were consistently down-regulated during hibernation including L19, L21, L36a, S17, S12 and S29 (Eddy & Storey, 2002). This further implicates control of the ribosomes as critical to the inhibition of protein synthesis in hibernation. One relevant issue to all uses of cDNA arrays in comparative animal systems is that of heterologous probing – the use of arrays containing immobilized cDNAs from one species to screen the mRNA populations of another species. Clearly, there are gene sequence differences between species that could prevent the cDNA probes synthesized from the mRNA of the test species from hybridizing with the bound cDNA fragments on the arrays so that a certain percentage of the genes on the array will not cross-react. This is a problem if the goal of the screening is to evaluate the responses of specific genes to the stress but if the goal is instead to screen broadly to find new stress-responsive genes, then the positive ‘hits ’ from cDNA array screening can easily provide a researcher with months or years of follow-up work to investigate the roles of each of these genes in hypometabolism. In our experience, heterologous probing works very well for mammalian hibernators. In our first studies with rat macroarrays we found that the percentage of cDNAs that cross-reacted was 93% for S. tridecemlineatus and 73% for the little brown bat, Myotis lucifugus (Eddy & Storey, 2002). Using the human 19K microarrays, the initial results were poor with a cross-reaction rate of only 15–20 % but we were able raise this to 85–90% using a simple reduction in the post-hybridization washing temperature from 50 xC, as suggested by the manufacturer, to room temperature (21 xC) (Eddy & Storey, 2002). We have also used human 19K cDNA arrays to screen for anoxia-induced changes in gene expression in hepatopancreas of L. littorea. Not unexpectedly considering the phylogenetic distance between gastropods and humans, cross-reactivity was low at only 18.35 %. Of those genes that did show significant cross-reaction due to high sequence identity, most showed no change in transcript levels in anoxia (88.8 %) and only 0.6 % showed reduced transcripts ; these data are in agreement with the idea that mRNA content is largely preserved during hypometabolism. However, 10.6 % of genes on the array were putatively up-regulated by twofold or more during anoxia. This represented over 300 genes and indicates scope for a widespread response to anoxia by many cellular functions in this facultative anaerobe. The genes identified included protein phosphatases and kinases, mitogen-activated protein kinase interacting factors, translation factors, antioxidant enzymes, Metabolic rate depression in animals and nuclear receptors (Larade & Storey, 2002 a). Not all of these candidate genes will be confirmed as anoxia upregulated when studied in detail using homologous probes and techniques such as Northern blotting or quantitative RT-PCR, but nonetheless, the results are sure to provide several new leads to follow. The use of cDNA array screening will certainly lead to novel discoveries about any system to which it is applied but a few caveats need mentioning. Apart from the sometimes low cross-reactivity with heterologous probing, array screening may still be limited in its ability to detect transcripts that are in low copy number or to confirm significant differences for changes of less than twofold. Furthermore, changes in mRNA transcript levels do not always imply changes of equal magnitude in protein levels or in the impact on metabolism. Protein levels are influenced by several additional layers of control including post-transcriptional processing of mRNA, differential mRNA stability, selective translation of transcripts by ribosomes, etc. The impact of altered gene expression on metabolism will also vary from protein to protein. For example, small changes in transcript and protein levels of a regulatory enzyme may have a much greater impact on metabolism than a much greater upregulation of a high activity equilibrium enzyme. (3 ) Hypometabolism-induced gene expression Despite the strong overall suppression of protein synthesis in hypometabolic states, examples of the specific up-regulation of selected genes and synthesis of specific proteins can be found in virtually every hypometabolic system that has been examined. For example, the gene for riboflavin binding protein was up-regulated during aestivation in liver of spadefoot toads Scaphiopus couchii and may contribute to vitamin storage during the months of dormancy (Storey, Dent & Storey, 1999). Various genes are up-regulated during insect or nematode diapause (e.g. Flannagan et al., 1998; Cherkasova et al., 2000; Denlinger, 2001; Moribe et al., 2001). For example, heat shock protein 70 was strongly upregulated at the initiation of diapause and persisted throughout 55 days of diapause, but disappeared within 12 h when diapause was terminated in pupae of the flesh fly Sarcophaga crassipalpis (Rinehart, Yocum & Denlinger, 2000). Gene upregulation during hypometabolism in hibernating mammals and anoxia-tolerant animals is summarized in the following sections. (4 ) Hibernation-induced gene expression Several groups are studying hibernation-responsive gene expression using multiple techniques and several animal and organ models. To date, one consistent finding from these studies is that entry into (and arousal from) hibernation apparently occurs with very few changes in gene expression. Thus, screening of a cDNA library made from heart of hibernating S. lateralis retrieved only two clones that were confirmed as up-regulated (Fahlman et al., 2000) whereas screening of a bat (M. lucifugus) skeletal muscle library found approximately 60 candidate clones but none that could be confirmed as up-regulated (Eddy & Storey, 2001). Use 223 of differential display PCR similarly failed to find hibernation-responsive genes in S. lateralis muscle (Eddy & Storey, 2001) and cDNA array screening showed low numbers of putatively up-regulated genes in ground squirrel and bat tissues (Eddy & Storey, 2002). O’Hara et al. (1999) found 29 candidate cDNAs that appeared to differ between euthermic and hibernating states in S. lateralis brain out of 4500 PCR products compared by differential display but none showed significant changes when examined with Northern blots. The apparent lack of major changes in gene expression during hibernation that is illustrated by these studies is not unexpected because (a) an energy-limited torpid state is not a time for major metabolic reorganization, and (b) cells and organs must remain competent to resume normal body functions rapidly during interbout arousals. However, these results also mean that the control of hibernation may be vested in only a handful of critical gene/ protein changes. To date, the genes that are known to be hibernation-responsive in ground squirrels include a2-macroglobulin in liver (Srere et al., 1995), moesin in intestine (Gorham, Bretscher & Carey, 1998), isozyme 4 of pyruvate dehydrogenase kinase (PDK) and pancreatic lipase in heart (Andrews et al., 1998 ; Buck, Squire & Andrews, 2002), isoforms of uncoupling protein and FABP in multiple tissues (Boyer et al., 1998 ; Hittel & Storey, 2001), the ventricular isoform of myosin light chain 1 (MLC1v) in heart and skeletal muscle (Fahlman et al., 2000), organic cation transporter 2 in kidney (Hittel & Storey, 2002b), the melatonin receptor (Mel1A) (McCarron et al., 2001) and four genes on the mitochondrial genome : NADH ubiquinone-oxidoreductase subunit 2 (ND2), cytochrome c oxidase subunit 1 (COX1) and ATPase subunits 6 and 8 (Fahlman et al., 2000; Hittel & Storey, 2002 a). Given that the mitochondrial genome is transcribed as a unit, the identification of the mitochondria-encoded genes suggests that a general up-regulation of the mitochondrial genome occurs during hibernation. This is particularly significant because transcript levels of related nuclear-encoded genes (e.g. subunit 4 of COX and ATPa, a nuclear encoded subunit of the mitochondrial ATP synthase) did not change during hibernation (Fig. 7) (Hittel & Storey, 2002 a). Interestingly, up-regulation of mitochondrial genes also occurs in freeze-tolerant and anoxia-tolerant animals (Storey, 1999; see Section V.5 below) so this may be a generalized response by stress-tolerant animals that requires further investigation. Other gene expression changes have been reported in brain of hibernating ground squirrels ; a 98 kDa protein with a phosphotyrosine moiety was found in membrane fractions during a hibernation bout but disappeared within 1 h of arousal (Ohtsuki et al., 1998) and increased expression of ‘ intermediate-early’ genes that code for selected transcription factors (c-fos, junB, c-Jun) occurred during late torpor and peaked during arousal (O’Hara et al., 1999). Notably, a substantial number of hibernation-responsive genes have been found in heart (Fig. 7). This may be related to the fact that the heart must continue to work during torpor and adjustments in gene expression are undoubtedly necessary to optimize cardiac muscle function with respect to the changes in temperature, work load and fuel availability that occur in torpor. Kenneth B. Storey and Janet M. Storey 224 torpor. For example, up-regulation of a2-macroglobulin during hibernation drew attention to the clotting cascade in hibernators. a2-Macroglobulin is a protease inhibitor that binds and inhibits several of the proteases that catalyze steps in the clotting cascade, including thrombin which cleaves fibrinogen to release fibrin monomers that polymerize into a fibrin clot. Increased a2-macroglobulin synthesis and export by liver (Srere et al., 1995), along with reduced platelet numbers and other adjustments to components of the clotting cascade, contribute to the observed decrease in clotting capacity of blood during torpor, an adaptation that minimizes spontaneous clot formation in the microvasculature under the low blood flow (ischemic) conditions of the hibernating state (Drew et al., 2001). 7 Ratio hibernating: euthermic Heart genes 6 5 4 3 2 1 AT P– a AT P6 /8 Co x4 Co x1 FA BP –A N ad 2 FA BP –H M LC 1v 0 Fig. 7. Histograms showing the effect of hibernation on gene up-regulation in ground squirrel heart. Relative transcript levels (determined from Northern blots) in hibernating versus euthermic heart are shown for eight genes : MlC1v, myosin light chain 1 ventricular isoform ; Nad2, subunit 2 of NADH-ubiquinone oxidoreductase ; FABP-H and FABP-A, heart and adipose isoforms of fatty acid binding protein, respectively ; Cox1 and Cox4, subunits 1 and 4 of cytochrome c oxidase ; ATP6/8, ATPase 6/8 bicistronic mRNA ; and ATPa, alpha subunit of the mitochondrial ATP synthase. Nad2, Cox1 and ATP6/8 are mitochondria-encoded subunits and Cox4 and ATPa are nuclear-encoded subunits of mitochondrial proteins. Data are means¡SEM, N=3. Data on MLC1v and Nad2 transcripts are from Spermophilus lateralis (Fahlman et al., 2000) ; all others are from S. tridecemlineatus (Hittel & Storey, 2001, 2002a). Some proteins are also down-regulated during hibernation (Kondo & Kondo, 1992 ; Soukri et al., 1996; Schmidt & Kelley, 2001). Particularly interesting is the suppression of insulin-like growth factor and of its plasma binding protein (IGFBP-3) during hibernation (Schmidt & Kelley, 2001). The growth-regulatory IGF axis controls energy-expensive somatic growth in skeleto-muscular and other tissues and its suppression during hibernation could be a key contributor to the hypometabolic state. Prostaglandin D2 synthase activity was reduced in late torpor in hypothalamus of S. lateralis but rebounded during arousal (O’Hara et al., 1999). Four proteins in the blood of euthermic chipmunks (Tamius asiaticus) also disappeared when the animals were hibernating (Kondo & Kondo, 1992) ; one of these showed high homology with a1-antitrypsin. Interestingly, a1-antitrypsin and three other acute-phase blood proteins were unaffected during hibernation in S. richardsonii but levels of a fifth member of the family, a2-macroglobulin, increased significantly (Srere et al., 1995). Results from both species suggest modification of the acute phase response to injury and infection during hibernation. (a ) a2-Macroglobulin The known hibernation-specific gene responses suggest some of the metabolic adjustments that are needed to support ( b ) PDK4 PDK4 expression during hibernation has a role in the control of fuel selection for the torpid animal (Andrews et al., 1998; Buck et al., 2002). Pyruvate dehydrogenase (PDH) is the entry point for carbohydrate into the tricarboxylic acid cycle. During hibernation, most organs switch to an almost exclusive dependence on lipid oxidation for energy generation in order to spare carbohydrate for selected tissues such as brain. A key enzyme in the control of carbohydrate catabolism is PDH and the amount of active PDH is strongly reduced during hibernation or daily torpor (Storey, 1997; Heldmaier et al., 1999), the mechanism being PDKmediated phosphorylation. Isozyme 4 of PDK is strongly expressed in heart and skeletal muscle of mammals and is responsive to physiological stresses including starvation and diabetes (Buck et al., 2002). PDK4 transcripts and protein were strongly induced in heart, skeletal muscle and white adipose tissue of ground squirrels during the winter with high levels maintained during both hibernation and interbout arousals (Buck et al., 2002). High PDK4 levels during the winter would keep PDH in an inactive state, thereby minimizing carbohydrate catabolism by muscles or carbohydrate use as a substrate for lipogenesis by adipose tissue. ( c ) Myosin Gene expression studies also give evidence of myosin restructuring in support of low-temperature cardiac function in hibernators. Myosin is made up of two heavy chains (MHC) and four light chains (MLC) that are classified as either alkali (MLC1, MLC3) or regulatory (MLC2) light chains ; the latter are subject to reversible phosphorylation. Transcript levels of the ventricular isoform of MLC1 rose 2– 3-fold during hibernation in S. tridecemlineatus heart and skeletal muscle (Fig. 7) implicating an increased content of this subunit in the myosin motor at low temperature (Fahlman et al., 2000). Other changes occurred in hamster (Cricetus cricetus) heart; the phosphorylation state of MLC2 decreased from 45% in summer to 23% in torpor and the proportions of MHC isoforms changed from a dominance of the b isoform (79% of total) in summer hamsters and winter-active animals at 22 xC to near-equal amounts of the Metabolic rate depression in animals a (53%) and b (47%) forms in hibernators (Morano et al., 1992). Changes in the expression of myosin genes and in the mix of myosin isoforms represented in a muscle is a well-known response to various stimuli (stretch, electrical stimulation, work load) in mammals and to temperature change in fish and is key to adapting the myosin motor to optimize contractile properties for different demands placed on the muscle (Goldspink & Yang, 2001). It is not surprising, then, that myosin restructuring occurs during hibernation in heart. During hibernation, the heart continues to beat but at a much lower body temperature and with a much lower rate than normal. However, peripheral resistance increases substantially and to compensate for this the force of contraction actually increases (Wang, 1989). Changes in sarcoplasmic reticulum Ca2+ storage and release have been tested as a possible reason for enhanced contractility in hibernation (Wang & Lee, 1996) but a change in the composition of muscle proteins would also provide an optimal mix of isoforms to adapt the contractile apparatus to the new work load and thermal conditions of the torpid state. 225 for triglyceride delivery via the blood. Hence, expression of A-FABP in hibernator heart provides the organ with access to both intracellular and extracellular lipid reserves for fuel. Analysis of the sequence of H-FABP from ground squirrels also showed three unique amino acid substitutions that place polar amino acids in positions previously filled by nonpolar or hydrophobic amino acids (Hittel & Storey, 2001). The substitutions occur in positions that would alter the flexibility of the protein and may contribute to the effective function of H-FABP at low temperatures. Studies with the liver isoform of FABP show that fatty acid binding by ground squirrel L-FABP is temperature insensitive over the range 5–37 xC whereas kinetic properties of rat L-FABP are compromised at low temperature (Stewart, English & Storey, 1998). Amino acid substitutions to H-FABP may similarly create temperature insensitive properties that allow the protein to function optimally in fatty acid transport over a wide range of body temperatures. ( e) Uncoupling proteins ( d ) Fatty acid binding proteins An up-regulation of the genes for heart (H) and adipose (A) isoforms of FABP during hibernation in BAT and heart of ground squirrels and the confirmation of enhanced synthesis of FABP protein in BAT during hibernation (FABP mRNA is in the heavy polysome fraction, Western blots show increased protein content) emphasizes the importance of lipid oxidation in these organs during hibernation (Figs 6 and 7) (Hittel & Storey, 2001, 2002 b). Heart switches to an almost total reliance on lipid oxidation during torpor and lipidfueled thermogenesis by uncoupled mitochondria in BAT is critical for rewarming the hibernator during arousal. The occurrence of both isoforms in both tissues is unusual but can be explained by their different functions in fatty acid transport. The A isoform of FABP is believed to carry fatty acids to and from intracellular lipid droplets and because it can form a complex with hormone-sensitive lipase, a prime function seems to be to carry fatty acids away from intracellular lipid droplets after triglyceride hydrolysis and to the mitochondria for oxidation (Vogel-Hertzel & Bernlohr, 2000). By contrast, the role of H-FABP is to pick up incoming fatty acids at the plasma membrane and transport them to the mitochondria. The presence of both isoforms in BAT reflects the fact that the tissue uses both its own internal lipid reserves and fatty acids imported from white adipose tissue to fuel nonshivering thermogenesis. Vertebrate heart typically displays only the H-isoform because it imports its fatty acids and induction of A-FABP in heart of hibernating ground squirrels (transcripts were not found in euthermic heart) is a first in the mammalian literature. Its presence could serve two functions : (1) because A-FABP also occurs in the hearts of Antarctic teleost fishes (Vayada et al., 1998), the presence of A-FABP may be important for low-temperature function, or (2) hearts of hibernating ground squirrels maintain substantial intracellular triglyceride lipid droplets (Burlington et al., 1972) that are probably needed to meet the demand for high rates of fatty acid oxidation during arousal that could exceed the capacity Hibernation-associated changes in the expression of uncoupling protein (UCP) isoforms also occur (Boyer et al., 1998). High rates of fatty acid oxidation fuel thermogenesis in hibernator organs both to support a minimum body temperature during torpor and to reheat the body rapidly during arousal. Heat production comes from nonshivering thermogenesis in BAT and also from shivering in skeletal muscles. Nonshivering thermogenesis in BAT involves the uncoupling protein UCP1 that dissipates the proton motive force across the inner mitochondrial membrane to release energy as heat rather than trapping it in the synthesis of ATP. The amount and activity of UCP1 in rodent BAT is strongly stimulated by cold exposure (Nizielski, Billington & Levine, 1995). Recent studies have explored the expression of UCP1 and its two homologues in hibernators (Boyer et al., 1998 ; Praun et al., 2001). UCP2 is expressed in multiple tissues and UCP3 occurs mainly in skeletal muscles. Increased mRNA levels for all three isoforms were found in BAT during cold exposure in hamsters but UCP2 and UCP3 mRNA levels in muscle did not change (Praun et al., 2001). By contrast, during hibernation in ground squirrels, UCP2 mRNA levels increased in white adipose tissue by 1.6-fold and UCP3 mRNA rose by threefold in skeletal muscle whereas UCP1 mRNA was not affected in BAT when hibernation was at ambient temperatures above 0 xC (Boyer et al., 1998). However, when thermogenic demands were increased by lowering the ambient temperature to subzero values, UCP1 mRNA increased approximately twofold in BAT compared with transcript levels in animals hibernating above 0 xC ; UCP2 mRNA in white adipose and UCP3 mRNA in skeletal muscle also rose. Although the thermogenic capacity of BAT was believed to be fully developed before hibernation begins, these results show that the torpid animal still has a capacity to respond with further UCP1 expression if thermal stress during hibernation is severe. Furthermore, the results for UCP2 and UCP3 suggest that tissues other than BAT may contribute to nonshivering thermogenesis in the hibernator. 226 (5 ) Anoxia-induced gene expression The anoxic state is an energy-limited one in which energyexpensive processes such as protein synthesis are restricted by mechanisms discussed earlier. Nonetheless, selected gene expression continues and various examples of anoxiastimulated up-regulation or induction of genes are now known, presumably representing genes whose protein products play key roles in anaerobic metabolism or in protecting cells from damage. (a ) Gene expression during anoxia in turtles We first explored anoxia-induced gene expression in the turtle, Trachemys scripta elegans, a widely studied model species that can endure total oxygen deprivation for as long as 3–4 months when submerged in cold water with a metabolic rate only approximately 10 % of the corresponding aerobic rate ( Jackson, 2001). Differential screening of a cDNA library made from heart of anoxia-exposed adult turtles (20 h submerged in N2-bubbled water at 7 xC) identified the anoxic up-regulation of three genes coded on the mitochondrial genome : Cox1 that encodes cytochrome C oxidase subunit 1 (COX1), Nad5 that encodes subunit 5 of NADH-ubiquinone oxidoreductase (ND5), and the mitochondrial WANCY (tryptophan, alanine, asparagine, cysteine and tyrosine) tRNA gene cluster (Cai & Storey, 1996, 1997). All three transcripts showed a similar pattern of response ; within 1 h of anoxia exposure transcript levels of Cox1, Nad5 and WANCY in heart had risen by 4.5-, 3- and 3.5-fold, respectively (Cai & Storey, 1996, 1997). Levels remained high over a 20 h anoxia excursion and then declined again during aerobic recovery. Cox1 and Nad5 were also upregulated in red muscle, brain and kidney during anoxia. A subsequent study revealed two other mitochondrially encoded genes that were up-regulated in liver of anoxic turtles : Cytb, encoding the protein cytochrome b and Nad4 which codes for subunit 4 of ND (Willmore, English & Storey, 2001). Cytb transcript levels were 5.2–5.7-fold higher than control values over the entire anoxia exposure (1–20 h), decreased by about half within 1 h of aerobic recovery and fell to near control values by 5 h recovery. Cytb transcripts also increased by threefold in anoxic kidney. Nad4 transcripts rose by 13-fold in liver within 1 h of anoxia exposure and then declined to 5–6-fold higher than controls during longer term anoxia. Mitochondrial genes are also up-regulated in several other instances of low oxygen stress. Freezing is an ischemic stress that cuts off oxygen delivery to tissues when plasma freezes. In response to freezing Cox1 and Nad5 were upregulated in freeze-tolerant turtles Chrysemys picta (Cai & Storey, 1996). Nad4 transcripts rose in liver of freeze-tolerant wood frogs Rana sylvatica and transcripts for subunits 6 and 8 of the F0F1ATPase complex increased in frog brain during freezing (S. Wu & K. B. Storey, unpublished observations). Transcripts of Cox2, another mitochondrially encoded protein subunit, rose 6–7-fold during anoxia in L. littorea (K. Larade, unpublished data) and, as mentioned previously, four genes on the mitochondrial genome (Nad2, Cox1 and ATPase subunits 6 and 8) were also up-regulated during Kenneth B. Storey and Janet M. Storey mammalian hibernation (Fahlman et al., 2000; Hittel & Storey, 2002 a). Hibernation is a situation of greatly reduced blood flow that is being used as a model for ischemia resistance in mammals (Frerichs et al., 1994 ; Bell et al., 2002). All of these proteins are large complexes containing multiple subunits coded on both the nuclear and mitochondrial genomes (e.g. three of the 13 subunits of COX1 are on the mitochondrial genome and six of the 41 subunits of ND) (Azzi, Muller & Labonia, 1989 ; Ohnishi, 1993) and, significantly, we have encountered no case to date where a nuclearencoded subunit of one of these proteins was identified as upregulated in the hypometabolic state (anoxia, frozen, hibernation). The question remains as to why mitochondrial transcript levels increase under hypometabolic conditions associated with anoxic or ischemic stress. Mitochondrial DNA contains only one promoter on each of the L and H strands and all genes are transcribed as one RNA precursor from the same initiation site (except for rRNA genes) (Gilham, 1994). The long polycistronic messages are then cleaved to give the individual RNA species. This mode of transcription can explain the parallel increases in Cox1, Nad5 and WANCY transcripts in anoxic turtle heart but more difficult to explain are the tissue-specific differences in gene up-regulation. For example, Cox1 and Nad5 transcripts rise in heart but not in liver during anoxia whereas Cytb transcripts rise in liver but not in heart (Cai & Storey, 1996; Willmore et al., 2001). An extensive analysis of the response to anoxia by all genes on the mitochondrial genome in multiple tissues of a single anoxia-tolerant species may resolve the question. ( b ) Gene expression during anoxia in marine snails Recent studies of anoxia-induced gene expression in the hepatopancreas of the marine gastropod, L. littorina, have documented the up-regulation of several genes including ribosomal protein L26, ferritin heavy chain, cytochrome c oxidase subunit II (COII), granulin/epithelin, a novel gene that we have named kvn and various unidentified genes (Larade et al., 2001; Larade, 2002; Larade & Storey, 2002a, b). Based on their known roles in other organisms, it is not difficult to infer functions for the protein products of some of these genes during anoxia. The gene encoding ribosomal protein L26 was upregulated during anoxia in L. littorea. Northern blots showed that levels of L26 transcripts rose to a maximum 4.7-fold increase in hepatopancreas after 96 h of anoxia exposure whereas a 3.4-fold increase was seen after 48 h in foot, compared with levels in aerobic snails (Fig. 3) (Larade et al., 2001). However, transcript levels decreased significantly within 1 h when snails were returned to aerobic conditions for recovery. Nuclear run-off assays confirmed that the elevated transcript levels were the result of an increased rate of L26 mRNA transcription ; 32P-dUTP incorporation was approximately twofold higher in nuclei isolated from hepatopancreas of 48 h anoxic snails than in nuclei from aerobic animals. The L26 protein resides at the interface of the large and small ribosomal subunit, apparently at or near the ribosomal A site where it is likely involved in subunit interactions (Lee & Horowitz, 1992). L26 can be Metabolic rate depression in animals 227 Relative transcript level 0 1 2 3 Control A B Anoxia Freezing db – cGMP a a a db – cAMP PMA Ca lonophore Fig. 8. Expression of mRNA transcripts encoding L26 ribosomal protein during in vitro incubation of Littorina littorea hepatopancreas samples under multiple conditions : anoxia (N2 bubbling at 4 xC for 12 h), freezing (at x7 xC for 12 h ), or incubation (at 4 xC for 2 h) with second messengers dibutyryl cyclic GMP, dibutyryl cyclic AMP, phorbol 12-myristate 13-acetate (PMA) or calcium ionophore (A23187). After incubation, total RNA was isolated, resolved on a formaldehyde gel, blotted onto nitrocellulose and hybridized with 32P-labeled L26 probe. The left column shows sample Northern blots for paired incubations of hepatopancreas samples under (A) normoxic and (B) experimental conditions. The bar graphs on the right show the scanned intensity of experimental samples relative to their paired normoxic controls ; data are means¡SEM, N=3. a, significantly different from control, P<0.05. cross-linked to elongation factor 2 (eEF-2), implicating it as a protein involved in forming the region that binds eEF-2 to the 60S ribosomal subunit preceding translocation of peptidyl-tRNA from the A to the P site during peptide bond formation (Nygard, Nilsson & Westermann, 1987). The link with eEF-2 is interesting because, as discussed earlier, inhibitory control of eEF-2 is one of the mechanisms that suppresses the rate of protein synthesis during hypometabolism. The function of anoxia-induced up-regulation of L26 mRNA levels remains elusive but it is possible that enhanced L26 protein levels during anoxia could contribute in some way to eEF-2 control. Alternatively, elevated L26 mRNA during anoxia might support a rapid synthesis of this protein once aerobic conditions were re-established, the production of L26 protein perhaps being key for the reestablishment of ribosomal function. Ribosome production is typically strictly coordinated with respect to the synthesis of component proteins (Mager, 1988) so future studies need to address the transcriptional and translational responses of other ribosomal components to anoxia. Anoxia exposure of L. littorea also stimulated a twofold increase in mRNA transcripts encoding the ferritin heavy chain in hepatopancreas; elevated levels were sustained throughout anoxia but fell within 1 h of oxygenated recovery (Larade, 2002). Western blotting confirmed an increase in protein content. Ferritin sequesters iron and, by doing so, plays an important role in antioxidant defence because free iron is a major catalyst in the production of oxygen free radicals (Orino et al., 2001). Anoxia-tolerant animals appear to avoid such oxidative damage by maintaining constitutively high levels of antioxidants and/or improving enzymatic antioxidant defences when stimulated by anoxic/ hypoxic conditions (Hermes-Lima, Storey & Storey, 1998; Pannunzio & Storey, 1998). Ferritin synthesis, providing enhanced iron storage, adds another layer of antioxidant defence. 228 A novel gene, kvn, was also up-regulated during anoxia in L. littorea hepatopancreas. The gene codes for a protein of 99 amino acids with a predicted molecular weight of 12 kDa (Larade & Storey, 2002b). Protein function is unknown but analysis of the putative amino acid sequence showed domains including casein kinase II phosphorylation sites and an N-myristoylation site as well as a 15 residue hydrophobic signal sequence at the N terminal. Such signal sequences generally direct proteins to the endoplasmic reticulum where they are processed and secreted to a final destination. Hence, KVN, which does not contain any known retention signals or localization motifs, is predicted to be an extracellular protein (Horton & Nakai, 1997). Levels of kvn transcripts increased gradually in hepatopancreas during anoxia, reaching a peak at 5.8-fold higher than control levels after 48 h anoxia but falling sharply within 1 h of oxygenated recovery. Nuclear run-off assays confirmed that the rise in kvn transcripts during anoxia was due to increased transcription, and analysis of kvn distribution on ribosomes showed a high proportion of kvn in the heavy polysome fraction (Larade & Storey, 2002b). Hence, it appears that kvn is actively translated during anoxia. Ongoing studies are seeking its function. (c ) cGMP mediation of anoxia-induced gene expression The regulatory controls on anoxia-induced gene expression in L. littorea are being explored using in vitro incubations of hepatopancreas explants (Larade et al., 2001 ; Larade, 2002; Larade & Storey, 2002 b). Transcript levels of L26, kvn and ferritin all increased when tissues were incubated in a medium bubbled with nitrogen gas as compared with aerobic control samples, confirming that anoxia stimulates gene up-regulation both in vivo and in vitro. Effects of incubation with stimulators of protein kinases (dibutyryl cAMP, dibutyryl cGMP, calcium ionophore A23187, and phorbol 12myristate 13-acetate) were then tested in aerobic incubations (Fig. 8). Transcript levels of all three genes were elevated when tissues were incubated with dibutyryl cGMP whereas only ferritin message responded to any of the other treatments. This implicates a cGMP-mediated signaling cascade in the gene expression response to anoxia in L. littorea and agrees with previous studies that have implicated cGMPdependent protein kinase (PKG) in the regulation of various metabolic responses to anoxia in marine molluscs. For example, PKG mediates the anoxia-induced phosphorylation of selected enzymes (e.g. pyruvate kinase) as part of glycolytic rate depression (Storey, 1993). Thus, it appears likely that the low oxygen signal that stimulates anaerobic adaptations and metabolic rate depression is transmitted via a cGMP and PKG signal transduction pathway in anoxia-tolerant molluscs. A well-known activator of guanylyl cyclases is the diffusible signal molecule, nitric oxide (NO) (Stamler, Singel & Loscalzo, 1992). In molluscs, intracellular cGMP levels have been shown to increase in the nervous system of various species due to activation of soluble guanylyl cyclase by NO and nitric oxide synthase activity has been confirmed in a large number of gastropod species (Moroz, 2000). Recent studies have shown that NO is involved in low oxygen signaling in Drosophila melanogaster Kenneth B. Storey and Janet M. Storey (Wingrove & O’Farrell, 1999) and this, coupled with the evidence cited above of cGMP mediation of anoxia-induced events in molluscs suggests that the NO/cGMP signaling pathway may be central in the response to oxygen deprivation in anoxia-tolerant species. VI. CONCLUSIONS 1. Metabolic arrest is critical for the survival of many species on Earth ; by suppressing metabolic rate to low levels, organisms can enter hypometabolic states that allow them to endure long-term exposure to stressful environmental conditions. 2. The biochemical regulatory mechanisms that control metabolic rate depression are broadly conserved across phylogenetic lines and include (a) the use of reversible protein phosphorylation to suppress the rates of major energy producing and energy consuming pathways to reach a new lower net rate of ATP turnover, (b) the selected upregulation of specific genes despite an overall suppression of transcription and translation, and (c) protection and stabilization of macromolecules to provide for a rapid return to normal life when environmental conditions allow. 3. Many avenues of research on hypometabolism remain to be explored, particularly the identification and role of various novel genes that are stress-induced and the mechanisms of signal transduction (hormones, receptors, intracellular messengers, transcription factors) that mediate the transition to the hypometabolic state. VII. 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